Compositions and Methods for Modulating PGC-1Alpha to Treat Neurological Diseases and Disorders

ABSTRACT

The present invention provides methods for modulating mitochondrial function, modulating lesion formation in the brain, modulating neurite growth, modulating neuronal degeneration, and treating and preventing neurological diseases or disorders comprising modulating the expression of activity of PGC-1α. The present invention also provides an animal, e.g., transgenic mouse, in which the PGC-1α gene is misexpressed. Methods for identifying compounds which are capable of treating or preventing a neurological disease or disorder are also described.

GOVERNMENT RIGHTS

This invention was made at least in part with support by grants awardedfrom the National Institute of Diabetes and Kidney Diseases (NIDDK) ofthe National Institutes of Health, grant numbers DK54477, DK61562, andK01DK065584. The U.S. government may have certain rights in theinvention.

BACKGROUND OF THE INVENTION

Understanding the regulatory circuits that govern cellular energy andglucose metabolism has been a focus of research interest in the pastdecade. Recent studies have implicated transcription coactivators of thePGC-1 family, in particular PGC-1α and PGC-1β, as important regulatorsof mitochondrial biogenesis and cellular respiration in several celltypes (Kelly, D. P., and Scarpulla, R. C. (2004) Genes Dev 18, 357-368;Puigserver, P., and Spiegelman, B. M. (2003) Endocr Rev 24, 78-90).Notably, the expression of PGC-1α has been found to be dysregulated indiabetic liver and skeletal muscle, tissues critical for maintainingnormal blood glucose levels, while PGC-1β mRNA levels are also loweredin diabetic muscle (Mootha et al. (2003) Nat Genet. 34, 267-273; Patti,M. et al. (2003) Proc Natl Acad Sci USA 100, 8466-8471; Yoon J. C., etal. (2001) Nature 413, 131-138). PGC-1α was initially identified as acold-inducible coactivator for PPARγ in brown fat (Puigserver et al.(1998) Cell 92, 829-839). Subsequent studies revealed that PGC-1α isable to bind to and augment transcriptional activities of many nuclearreceptors and several other transcription factors outside the nuclearreceptor superfamily. Adenoviral-mediated or transgenic expression ofPGC-1α in cultured cells and in vivo leads to activation ofmitochondrial biogenesis and increases in cellular respiration (Lehmanet al., (2000) J Clin Invest 106, 847-856; Lin et al. (2002b) Nature418, 797-801; St-Pierre et al. (2003) J Biol Chem 278, 26597-26603; Wuet al. (1999) Cell 98, 115-124). Consistent with a regulatory role inthe cellular adaptations to increased energy requirements, theexpression of PGC-1α itself is highly regulated in response tonutritional and environmental stimuli. For example, PGC-1α mRNA isstrongly induced in brown fat by cold exposure and in skeletal musclefollowing physical activity (Baar et al. (2002) Faseb J 16, 1879-1886;Goto et al. (2000) Biochem Biophys Res Commun 274, 350-354; Puigserveret al. (1998) Cell 92, 829-839). Increased PGC-1α levels in thesetissues lead to enhanced mitochondrial electron transport activitiesthat enable cells to meet rising energy demands, such as during adaptivethermogenesis in brown fat and contraction in muscle.

In addition to its role in mitochondrial biology, PGC-1α also regulatesseveral key metabolic programs that go beyond simple mitochondrialbiogenesis and oxidative phosphorylation. For example, PGC-1α drivesexpression of myofibrillar proteins characteristic of slow-twitch musclefibers when expressed in fast-twitch muscle beds of transgenic mice (Linet al. (2002b) Nature 418, 797-801). In the liver, PGC-1α mRNA level israpidly induced following short-term fasting (Yoon et al. (2001) Nature413, 131-138). Adenoviral-mediated expression of PGC-1α in culturedprimary hepatocytes and in live rats leads to activation of the entireprogram of gluconeogenesis and increased glucose production (Yoon et al.(2001) Nature 413, 131-138). In all of these cases, PGC-1α interactswith cell-selective transcription factors to execute thesetissue-specific functions, such as MEF2c in skeletal muscle and HNF4αand FOXO1 in the liver.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatmodulation of PGC-1α, e.g., PGC-1α expression or activity, leads to themodulation of lesion formation, e.g., brain lesion formation,neurological degeneration, and neurite formation. Therefore, in oneaspect, the present invention provides a method for treating and/orpreventing a neurological disease or disorder in a subject, e.g., ahuman, by administering a PGC-1α modulator.

Examples of neurological diseases or disorders include Alzheimer'sdisease, Parkinson's disease, Huntington's disease, Pick's disease,Kuf's disease, Lewy body disease, neurofibrillary tangles, Rosenthalfibers, Mallory's hyaline, senile dementia, myasthenia gravis, Gilles dela Tourette's syndrome, multiple sclerosis (MS), amyotrophic lateralsclerosis (ALS), progressive supranuclear palsy (PSP), epilepsy,Creutzfeldt-Jakob disease, deafness-dytonia syndrome, Leigh syndrome,Leber hereditary optic neuropathy (LHON), parkinsonism, dystonia, motorneuron disease, neuropathy-ataxia and retinitis pimentosa (NARP),maternal inherited Leigh syndrome (MILS), Friedreich ataxia, hereditaryspastic paraplegia, Mohr-Tranebjaerg syndrome, Wilson disease, sporaticAlzheimer's disease, sporadic amyotrophic lateral sclerosis, sporadicParkinson's disease, autonomic function disorders, hypertension, sleepdisorders, neuropsychiatric disorders, depression, schizophrenia,schizoaffective disorder, korsakoff's psychosis, mania, anxietydisorders, phobic disorder, learning or memory disorders, amnesia orage-related memory loss, attention deficit disorder, dysthymic disorder,major depressive disorder, obsessive-compulsive disorder, psychoactivesubstance use disorders, panic disorder, bipolar affective disorder,severe bipolar affective (mood) disorder (BP-1), migraines,hyperactivity and movement disorders.

In one embodiment, a PGC-1α modulator is used in the methods of theinvention,

wherein the modulator is capable of modulating PGC-1α polypeptideactivity. In another embodiment, the modulator is a PGC-1α polypeptidecomprising the amino acid sequence of SEQ ID NO: 2, or a fragmentthereof. In still another embodiment, the modulator includes a PGC-1αpolypeptide comprising an amino acid sequence which is at least 90percent identical to the amino acid sequence of SEQ ID NO: 2.

In yet another embodiment, the PGC-1α modulator is an isolated naturallyoccurring allelic variant of a polypeptide consisting of the amino acidsequence of SEQ ID NO:2, wherein the polypeptide is encoded by a nucleicacid molecule which hybridizes to a complement of a nucleic acidmolecule consisting of SEQ ID NO:1 at 6×SSC at 45° C., followed by oneor more washes in 0.2×SSC, 0.1% SDS at 65° C.

In still a further embodiment, the PGC-1α modulator is capable ofmodulating PGC-1α nucleic acid expression. For example, the PGC-1αmodulator includes a PGC-1α nucleic acid molecule, e.g., a PGC-1αnucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,or a fragment thereof. In another embodiment, the PGC-1α modulator is amodulator of a transcriptional activator which modulates the expressionof PGC-1α.

In yet another embodiment, the PGC-1α modulator modulates mitochondrialfunction, e.g., mitochondrial function in the brain. In still anotherembodiment, the PGC-1α modulator is capable of modulating lesionformation in the brain. In still a further embodiment, the PGC-1αmodulator is capable of modulating neurite growth.

In another aspect, the invention provides a method of modulating brainlesion formation by contacting a cell with a PGC-1α modulator such thatbrain lesion formation is modulated. In yet another aspect, theinvention provides a method for modulating neuronal degeneration bycontacting a cell with a PGC-1α modulator such that neuronaldegeneration is modulated.

In still another aspect, the invention provides methods for identifyinga compound capable of treating or preventing a neurological disease ordisorder comprising the step of assaying the ability of the compound tomodulate PGC-1α nucleic acid expression or PGC-1α polypeptide activity.In one embodiment, a modulating compound is identified by detectingmodulation of mitochondrial function or by detecting modulation in theexpression or activity of mitochondrial genes, e.g., LDH2, Ndufb5,COX6a1, and ATP5j. In another embodiment, a modulating compound isidentified by detecting modulation in the expression or activity ofneuronal genes, e.g., NF—H, NF-M, MOBP, ATPa1, and ATP1a2. A PGC-1αmodulator identified by the methods of the invention includes, but isnot limited to, a small molecule, a nucleic acid molecule, apolypeptide, a peptide or peptidomimetic.

In still another aspect, the invention provides methods for assessingwhether a subject is afflicted with a neurological disease or disorderor is at risk of developing a neurological disease or disorder,comprising the step of detecting the expression of the PGC-1α gene orthe activity of PGC-1α in a cell or tissue sample of a subject, e.g.,cerebrospinal fluid, spinal fluid, and neural tissue.

In yet another aspect, the invention provides a non-human animal, inwhich a PGC-1α gene is misexpressed, e.g., a transgenic animal, inparticular, a mouse. In a further embodiment, the animal has a PGC-1αgene that is disrupted by the removal of DNA encoding all or part of thePGC-1α gene. The present invention also includes animals that arehomozygous for the disrupted gene or heterozygous for the disruptedgene. In one embodiment, the invention provides a transgenic mouse witha disruption of the PGC-1α gene, e.g., an insertion or deletion of thePGC-1α gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-F depict the generation of PGC-1α deficient mice. Inparticular, FIG. 1A depicts hybridization analysis of PGC-1α mRNA inliver and skeletal muscle using a probe spanning exons 3 to 5 of PGC-1α.Hybridization with a probe specific for ribosomal protein 36B4 wasincluded as a loading control. FIG. 1B depicts the results ofimmunoblotting of PGC-1α protein. Lysates containing in vitro translatedPGC-1α were used as a positive control. Note the absence of PGC-1αprotein in brown fat extracts from PGC-1α^(−/−) mice. FIGS. 1C-D depictH&E staining of paraffin-embedded liver sections. FIGS. 1E-F depict H&Estaining of plastic-embedded brown fat sections.

FIGS. 2A-E depict impaired glucose homeostasis and hepatic energymetabolism in the absence of PGC-1α. In particular, FIG. 2A depictsplasma glucose levels in PGC-1α^(+/+) (filled box), PGC-1α^(+/−) (dottedbox) and PGC-1α^(−/−) (open box) in the fed and fasted (24 hours)states. *p=0.0007. FIG. 2B depicts plasma insulin concentrations in thesame group of mice used in (2A). *p=0.005. FIG. 2C depicts total anduncoupled respiration in wild-type (+/+) and PGC-1α deficient (−/−)hepatocytes. *p<0.02. Data in FIGS. 2A-C represent mean±s.e.m.Furthermore, FIG. 2D depicts defective hormone-induced gluconeogenicgene expression in hepatocytes lacking PGC-1α. Primary hepatocytes wereisolated from PGC-1α^(+/+) and PGC-1α^(−/−) mice and treated with 0.2 μMor 1.0 μM of a combination of forskolin and dex for 3 or 6 hours beforeRNA isolation and hybridization. Hybridization for ribosomal protein36B4 mRNA was included as a loading control. FIG. 2E depicts the resultsof pyruvate tolerance test. Three-month old male mice were fastedovernight before receiving IP injection of a pyruvate solution asdescribed in the Examples Section. *p<0.0002; **p<0.02.

FIGS. 3A-E depict the constitutive activation of the gluconeogenicprogram in PGC-1α deficient liver. In particular, FIG. 3A depictshybridization analysis of mRNAs for metabolic genes in the fed andfasted liver. Three-month old male mice were fed ad libitum or fastedfor 24 hours before harvesting tissues for mRNA analysis. FIG. 3Bdepicts expression of mRNAs for transcription factors that regulatehepatic metabolism. Note the dramatic induction of C/EBPβ mRNA in fedPGC-1α^(−/−) liver compared to wild-type liver. FIG. 3C is a graphdepicting real-time PCR analysis of mRNA levels for the C/EBP familymembers. FIG. 3D is a graph which illustrates that PGC-1α does notcoactivate C/EBPβ. H2.35 hepatoma cells were transiently transfectedwith a UAS-luciferase reporter with Gal4-DBD-C/EBPβ in the presence orabsence of PGC-1α. Luciferase activity was measured 30 hours followingtransfection. FIG. 3E is a graph which depicts induction of endogenousgluconeogenic genes by C/EBPβ in primary hepatocytes. Primaryhepatocytes were isolated from PGC-1α^(+/+) (filled box) andPGC-1α^(−/−) (open box) mouse liver and infected with adenovirusesexpression GFP or C/EBPβ. Total RNA were harvested following 3 hours oftreatments with 0.2 μM forskolin and 0.1 μM dex. Relative abundance ofG6Pase and PEPCK mRNA was examined by real-time PCR followed bynormalization to 18S ribosomal RNA.

FIGS. 4A-F depict resistance to diet-induced obesity and insulinresistance in PGC-1α^(−/−) mice. FIG. 4A is a graph which depicts bodyweight of PGC-1α^(+/+) (filled circle, n=6) and PGC-1α^(−/−) (filledsquare, n=6) mice fed a high-fat diet. Twelve-week old males were fed ahigh-fat diet for 16 weeks. Body weight was measured weekly. FIG. 4Bdepicts representative DEXA scanning images of PGC-1α^(+/+) andPGC-1α^(−/−) mice after 12 weeks of high-fat feeding. FIG. 4C is a graphdepicting body fat content in PGC-1α^(+/+) and PGC-1α^(−/−) mice underchow (four month-old males) or high-fat feeding. Percent body fat wasdetermined by automated analysis of DEXA images with a program suppliedby manufacturer. *p=0.005; **p=0.001. FIG. 4D is a graph depictingfasting glucose and insulin levels in high-fat fed mice. *p=0.0001;**p=0.02. FIG. 4E is a graph depicting insulin tolerance test onhigh-fat fed PGC-1α^(+/+) (filled circle, n=5) and PGC-1α^(−/−) (filledsquare, n=5) mice. *p<0.004. FIG. 4F is a graph depicting glucosetolerance test in high-fat fed mice following an overnight fast.*p<0.02. Insulin and glucose tolerance tests were performed as describedin the Examples Section. The data in FIGS. 4A and 4C-F representmean±s.e.m.

FIGS. 5A-F are graphs depicting analysis of whole-body energy balanceand thermogenesis in PGC-1α^(−/−) mice. FIG. 5A illustrates that foodintake was measured in a two-day period and normalized to body weight.FIG. 5B illustrates whole body O₂ consumption in PGC-1α^(+/+) (n=5) andPGC-1α^(−/−) (n=5) mice as monitored by CLAMS. Shown is the averagedvalue over two days and three nights. *p<0.006. FIG. 5C depicts bodytemperature of 6 to 7-week old PGC-1α^(+/+) (circle, n=6) andPGC-1α^(−/−) (square, n=6) mice exposed to cold temperature (4° C.).*p<0.008. Data in FIGS. 5B-C represent mean±s.e.m. FIG. 5D illustratesgene expression in brown fat analyzed by quantitative real-time PCR.Intrascapular brown fat was dissected from PGC-1α^(+/+) (filled box) andPGC-1α^(−/−) (open box) mice maintained at 24° C. or after 5 hours ofcold exposure at 4° C. Primers specific for 18S ribosomal RNA were usedfor normalization. FIG. 5E depicts analysis of skeletal muscle geneexpression in wild-type (filled box) and PGC-1α deficient (open box)mice by quantitative real-time PCR. Quadriceps muscle was dissected from4-month old male mice and frozen at −80° C. before RNA isolation andanalysis. Primers specific for 18S ribosomal RNA were used fornormalization. FIG. 5F depicts the activation of AMPK in PGC-1α^(−/−)skeletal muscle. Tissue extracts were prepared from wild-type or PGC-1αnull quadriceps muscle and analyzed by immunoblotting using antibodiesspecific for phosphorylated AMPK (pAMPK) and ACC (pACC), or an antibodythat reacts with both phosphorylated and non-phosphorylated forms ofAMPK.

FIGS. 6A-D depict hyperactivity and limb clasping in PGC-1α^(−/−) mice.FIG. 6A depicts a representative trace of movement monitoring forPGC-1α^(+/+) (circles) and PGC-1α^(−/−) (squares) mice over a period ofthree days. FIG. 6B depicts a representative trace of whole body O₂consumption in PGC-1α^(+/+) (circles) and PGC-1α^(−/−) (squares) mice. Nand D denote night and day periods, respectively. FIG. 6C is a graphdepicting measurements of physical activity in 3-month old male micewith CLAMS. Shown is the average movement counts during the monitoringperiod. *p<0.01. FIG. 6D illustrates the limb clasping in PGC-1α^(−/−)mice.

FIGS. 7A-H depict the results of histological staining of brain sectionsfrom wild type and PGC-1α null mice. FIGS. 7A-B depict low magnificationpictures showing cortex (ctx) and striatum (STR) of PGC-1α^(+/+) mousebrain (FIG. 7A) and PGC-1α^(−/−) mice (FIG. 7B) stained with Luxol fastblue and H&E. Note spongiform pathology predominantly in the striatum ofthe PGC-1α^(−/−) mouse brain (red arrows). (Scalebar=200 μm). FIGS. 7C-Ddepict high magnification pictures of the striatum of wild type (FIG.7C) and PGC-1α^(−/−) brains (FIG. 7D) stained with Luxol fast blue/H&E.Shown are spongiform lesions in the striatum that are predominantlyassociated with the white matter. FIGS. 7E-F depicts immunohistochemicalstaining with an anti-GFAP antibody. Note abundant presence of reactiveastrocytes (blue arrows) in the striatum of PGC-1α^(−/−) mice (FIG. 7F),but not in wild type controls (FIG. 7E). FIGS. 7G-H depicts lessstriatal neurites are detected in the PGC-1α^(−/−) brain (FIG. 7H) witha neurofilament heavy chain antibody compared to wild-type striatum(FIG. 7G). (Scalebar=200 μm).

FIGS. 8 A-B depict gene expression analysis in mouse brain and neuritegrowth in cultured primary striatal neurons. FIG. 8A is a graphdepicting analysis of mitochondrial gene expression in wild-type (filledbox, n=4) and PGC-1α deficient (open box, n=6) mouse brain by real-timePCR. Whole brain was dissected from 3-month old male mice and frozen at−80° C. before RNA isolation and analysis. Primers specific for 18Sribosomal RNA were used for normalization. *p<0.02. FIG. 8B is a graphdepicting real-time PCR analysis of non-mitochondrial genes involved innormal brain function as in (FIG. A). *p<0.02.

FIGS. 9A-D depict genotyping of mice of the present invention by PCRwith tail DNA. In particular, FIG. 9A illustrates DNA that was isolatedfrom neomycin-resistant ES cell clones, digested with BamHI andsubjected to hybridization using probe L to detect homologousrecombination and the presence of the flox allele. FIGS. 9B-D illustratechimeric founders which were bred with wild-type C57/Bl6 mice to obtainoffspring containing a germ-line PGC-1α flox allele. Primers used forgenotyping include SEQ ID NO:s 41 and 42 for wild type animals and SEQID NO:s 43 and 44 for knock-out animals.

FIGS. 10A & B depict a model arising from these studies concerning thedietary control of gluconeogenesis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery thatmodulation of PGC-1α, e.g., PGC-1α expression or activity, leads to themodulation of lesion formation, e.g., brain lesion formation,neurological degeneration, and neurite formation. Therefore, in oneaspect, the present invention provides methods for treating and/orpreventing a neurological disease or disorder in a subject, e.g., ahuman subject, by administering to the subject a PGC-1α modulator, e.g.,a PGC-1α agonist or antagonist.

PGC-1α null animals, e.g., mice, have been generated which displayneurological defects, e.g., brain lesions, and histologicalabnormalities of the brain. The PGC-1α deficient animals also displaybehavioral abnormalities, e.g., hyperactivity, and an increase in energyexpenditure which correlates with their increased activity. Because ofthis increase in energy expenditure and hyperactivity, thePGC-1α-deficient animals are also resistant to obesity and insulinresistance.

It has been determined that the behavioral abnormalities of theseanimals are associated with lesions in the brain, e.g., the striatum, abrain area that plays a role in motor coordination. Without intending tobe bound by theory; the prominent spongiform lesions in the striatum andin other areas of the PGC-1α null mouse brain suggest that abnormal CNSfunction is likely underlying the hyperactivity in the null animals. Itis possible that many of these affected neurons play an inhibitoryfunction with respect to physical movements of the mice, in that theirloss is accompanied by increased movement and other neurologicalabnormalities. The PGC-1α-deficient animals also display alterations inthe expression of genes involved in oxidative metabolism and neuronalfunction, and display impaired neurite growth. Furthermore, thehyperactivity displayed by mice deficient in PGC-1α withneurodegeneration is similar to the hyperactivity associated withneurological diseases and disorders, including, for example,Huntington's disease (HD).

Accordingly, in one aspect, the present invention is based, at least inpart, on the discovery that PGC-1α functions in the development ofneurological diseases and disorders, including neurodegenerativediseases and disorders and movement disorders, and is involved inmitochondrial function in the brain, lesion formation in the brain,neurite generation, and neurological degeneration. Therefore, modulationof PGC-1α, e.g., modulation of the expression or activity of PGC-1αand/or the pathways controlled by PGC-1α, through genetic orpharmacological methods, can improve brain function in neurologicaldiseases and disorders or protect the brain from developingcharacteristics associated with neurological diseases or disorders, tothereby treat and/or prevent neurological diseases and disorders in asubject.

In another aspect, the present invention provides methods for modulatinga neurological disease or disorder in a subject by administering aPGC-1α modulator to induce PGC-1α expression or activity. The presentinvention also provides methods for modulating mitochondrial function,e.g., in the brain, in a subject by administering a PGC-1α modulator toinduce PGC-1α expression or activity. The present invention alsoprovides methods for modulating lesion formation and neurite growth byadministering a PGC-1α modulator to induce PGC-1α expression oractivity.

In another aspect, the invention features methods for identifying acompound which modulates the expression or activity of PGC-1α. Themethods include contacting PGC-1α or a cell expressing PGC-1α with atest compound and determining the effect of the test compound on theexpression or activity of PGC-1α to, thereby, identify a compound whichmodulates, e.g., increases or decreases, PGC-1α expression or activity.

In another aspect, the invention features a non-human animal, in whichthe gene encoding the PGC-1α protein is misexpressed. In preferredembodiments, the non-human animal is a transgenic animal. The transgenicnon-human animal can be, without limitation, a mammal; a bird; a reptileor an amphibian. Suitable mammals for uses described herein include,e.g., ruminants, ungulates, domesticated mammals, and dairy animals.Other suitable animals include goats, sheep, camels, cows, pigs, horses,oxen, llamas, chickens, geese, and turkeys. Methods for the preparationand use of such animals are known in the art. A protocol for theproduction of a transgenic pig can be found in White and Yannoutsos,Current Topics in Complement Research: 64th Forum in Immunology, pp.88-94; U.S. Pat. No. 5,523,226; U.S. Pat. No. 5,573,933; PCT ApplicationWO93/25071; and PCT Application WO95/04744. A protocol for theproduction of a transgenic rat can be found in Bader and Ganten,Clinical and Experimental Pharmacology and Physiology, Supp. 3:S81-S87,1996. A protocol for the production of a transgenic cow can be found inTransgenic Animal Technology, A Handbook, 1994, ed., Carl A. Pinkert,Academic Press, Inc. A protocol for the production of a transgenic sheepcan be found in Transgenic Animal Technology, A Handbook, 1994, ed.,Carl A. Pinkert, Academic Press, Inc.

DEFINITIONS

As used herein, the term “modulator of PGC-1α expression or activity”includes a compound or agent that is capable of modulating or regulatingPGC-1α expression or at least one PGC-1α activity, as described herein.A modulator of PGC-1α expression or activity can be an inducer of PGC-1αexpression or activity or an inhibitor of PGC-1α expression or activity.As used herein, an “inducer or agonist of PGC-1α activity” agonizes,stimulates, enhances, and/or mimics a PGC-1α activity, either completelyor partially. An “inducer or agonist of PGC-1α expression” increases,enhances, or stimulates PGC-1α expression, either completely orpartially, directly or indirectly. As used herein, an “inhibitor orantagonist of PGC-1α activity” antagonizes, reduces, or blocks PGC-1αactivity, either completely or partially. An “inhibitor or antagonist ofPGC-1α expression” reduces or blocks PGC-1α expression, eithercompletely or partially, directly or indirectly. Examples of PGC-1αinhibitors include small molecules, antisense PGC-1α nucleic acidmolecules, ribozymes, siRNA molecules, and anti-PGC-1α antibodies.Examples of PGC-1α inducers include PGC-1α mimetics, e.g.,peptidomimetics, small molecules, nucleic acid molecules encodingPGC-1α, and PGC-1α proteins or fragments thereof.

As used interchangeably herein, a “PGC-1α activity”, “biologicalactivity of PGC-1α” or “functional activity of PGC-1α” refers to anactivity exerted by a PGC-1α polypeptide or nucleic acid molecule on aPGC-1α responsive molecule, cell, or tissue, as determined in vitroand/or in vivo, according to standard techniques. In an exemplaryembodiment, a PGC-1α activity is the ability to modulate mitochondrialfunction, e.g., oxidative metabolism. In another embodiment, PGC-1αactivity is the ability to modulate the activity or expression of amitochondrial gene, e.g., LDH2,Ndufb5, COX6a1, or ATP5j. In yet anotherembodiment, a PGC-1α activity is the ability to modulate the expressionor activity of a neuronal gene, e.g., NF—H, NF-M, MOBP, ATPa1, orATP1a2. In a further embodiment, PGC-1α activity is the ability tomodulate lesion formation, e.g., brain lesion formation, in, forexample, the striatum. In yet another embodiment, PGC-1α activity is theability to modulate neurite formation and/or neuronal degeneration. Inanother embodiment, PGC-1α activity is the ability to modulate bodyweight and energy expenditure, e.g., via hyperactivity. In a furtherembodiment, PGC-1α activity is the ability to modulate gluconeogenesis,e.g., via coactivation of Foxo1, HNF4α, GR, and other factors. In stillanother embodiment, PGC-1α activity is the ability to modulate interactwith (e.g., bind to) nuclear hormone receptors. In a preferredembodiment, PGC-1α activity is the ability to modulate neurologicaldiseases or disorders, e.g., neurodegenerative diseases or disorders, ina subject.

As used herein, the term “neurological disease or disorder” includes anydisease, disorder, or condition which is caused by or related todysfunction or deficiency of the central nervous system, including, butnot limited to mitochondrial dysfunction, lesion formation, neuraldegeneration, or misregulation or modulation of any central nervoussystem specific pathway or central nervous system specific activity.Neurological diseases or disorders include neurodegenerative andcognitive disorders. Examples of neurological diseases and disordersinclude, but are not limited to, Alzheimer's disease, Parkinson'sdisease, Huntington's disease, Pick's disease, Kuf's disease, Lewy bodydisease, neurofibrillary tangles, Rosenthal fibers, Mallory's hyaline,senile dementia, myasthenia gravis, Gilles de la Tourette's syndrome,multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS),progressive supranuclear palsy (PSP), epilepsy, Creutzfeldt-Jakobdisease, deafness-dytonia syndrome, Leigh syndrome, Leber hereditaryoptic neuropathy (LHON), parkinsonism, dystonia, motor neuron disease,neuropathy-ataxia and retinitis pimentosa (NARP), maternal inheritedLeigh syndrome (MILS), Friedreich ataxia, hereditary spastic paraplegia,Mohr-Tranebjaerg syndrome, Wilson disease, sporatic Alzheimer's disease,sporadic amyotrophic lateral sclerosis, sporadic Parkinson's disease,autonomic function disorders, hypertension, sleep disorders,neuropsychiatric disorders, depression, schizophrenia, schizoaffectivedisorder, korsakoff's psychosis, mania, anxiety disorders, phobicdisorder, learning or memory disorders, amnesia or age-related memoryloss, attention deficit disorder, dysthymic disorder, major depressivedisorder, obsessive-compulsive disorder, psychoactive substance usedisorders, panic disorder, bipolar affective disorder, severe bipolaraffective (mood) disorder (BP-1), migraines, hyperactivity and movementdisorders. As used herein, the term “movement disorder” includesneurological diseases or disorders that involve the motor and movementsystems, resulting in a range of abnormalities that affect the speed,quality and ease of movement. Movement disorders are often caused by orrelated to abnormalities in brain structure and/or function. Movementdisorders include, but are not limited to (i) tremors: including, butnot limited to, the tremor associated with Parkinson's Disease,physiologic tremor, benign familial tremor, cerebellar tremor, rubraltremor, toxic tremor, metabolic tremor, and senile tremor; (ii) chorea,including, but not limited to, chorea associated with Huntington'sDisease, Wilson's Disease, ataxia telangiectasia, infection, drugingestion, or metabolic, vascular or endocrine etiology (e.g., choreagravidarum or thyrotoxicosis); (iii) ballism (defined herein as abruptlybeginning, repetitive, wide, flinging movements affecting predominantlythe proximal limb and girdle muscles); (iv) athetosis (defined herein asrelatively slow, twisting, writhing, snake-like movements and posturesinvolving the trunk, neck, face and extremities); (v) dystonia (definedherein as a movement disorder consisting of twisting, turning tonicskeletal muscle contractions, most, but not all of which are initiateddistally); (vi) paroxysmal choreoathetosis and tonic spasm; (vii) tics(defined herein as sudden, behaviorally related, irregular, stereotyped,repetitive movements of variable complexity); (viii) tardive dyskinesia;(ix) akathesia, (x) muscle rigidity, defined herein as resistance of amuscle to stretch; (xi) postural instability; (xii) bradykinesia; (xiii)difficulty in initiating movements; (xiv) muscle cramps; (xv)dyskinesias and (xvi) myoclonus.

The term “mitochondrial function” includes any cellular activity carriedout by mitochondria or mitochondrial genes, including mitochondria ormitochondrial genes in brain cells, e.g., neurons. For example,mitochondria play a role in a number of important cellular functionsincluding, for example, oxidative energy metabolism, amino acidbiosynthesis, fatty acid oxidation, steroid metabolism, and apoptosis.“Mitochondrial dysfunction” includes any failure or deficiency ofmitochondria or mitochondrial genes to carry out a mitochondrialfunction, e.g., oxidative energy metabolism.

The term “treatment”, as used herein, is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease or disorder, a symptom of a disease ordisorder or a predisposition toward a disease or disorder, with thepurpose of curing, healing, alleviating, relieving, altering, remedying,ameliorating, improving or affecting the disease or disorder, thesymptoms of disease or disorder or the predisposition toward a diseaseor disorder. A therapeutic agent includes, but is not limited to, smallmolecules, peptides, peptidomimetics, nucleic acid molecules,antibodies, ribozymes, siRNA molecules, and sense and antisenseoligonucleotides described herein.

As used herein, “administering a treatment to an animal or cell” isintended to refer to dispensing, delivering or applying a treatment toan animal or cell. In terms of the therapeutic agent, the term“administering” is intended to refer to contacting or dispensing,delivering or applying the therapeutic agent to an animal by anysuitable route for delivery of the therapeutic agent to the desiredlocation in the animal, including delivery by either the parenteral ororal route, intramuscular injection, subcutaneous/intradermal injection,intravenous injection, buccal administration, transdermal delivery andadministration by the intranasal or respiratory tract route.

As used herein, the term “compound” includes any agent, e.g., peptide,peptidomimetic, small molecule, or other drug, which binds to a PGC-1αprotein or has a stimulatory or inhibitory effect on, for example,PGC-1α expression or PGC-1α activity.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a gene,product, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter, e.g., a brain cell. PGC-1α may beexpressed in specific portions of the brain, e.g., in an animal model orin a subject to treat or prevent a neurological disease or disorder.Furthermore, in another embodiment, tissue specific PGC-1α knock-outanimal models may also be produced wherein PGC-1α is flanked (or“foxed”) by two or more lox sites, most commonly loxP sites, and isexcised using the Cre recombinase protein, as is known in the art anddescribed herein In one embodiment, a knock-out animal is used toevaluate the function of PGC-1α in a specific nervous system tissue.

The term “polymorphism” refers to the coexistence of more than one formof a gene or portion thereof. A portion of a gene of which there are atleast two different forms, i.e., two different nucleotide sequences, isreferred to as a “polymorphic region of a gene.” A polymorphic locus canbe a single nucleotide, the identity of which differs in the otheralleles. A polymorphic locus can also be more than one nucleotide long.The allelic form occurring most frequently in a selected population isoften referred to as the reference and/or wildtype form. Other allelicforms are typically designated, alternative, or variant alleles. Diploidorganisms may be homozygous or heterozygous for allelic forms. Adiallelic or biallelic polymorphism has two forms. A trialleleicpolymorphism has three forms.

The term “single nucleotide polymorphism” (SNP) refers to a polymorphicsite occupied by a single nucleotide, which is the site of variationbetween allelic sequences. A SNP usually arises due to substitution ofone nucleotide for another at the polymorphic site. SNPs can also arisefrom a deletion of a nucleotide or an insertion of a nucleotide relativeto a reference allele. Typically, the polymorphic site is occupied by abase other than the reference base. For example, where the referenceallele contains the base “T” (thymidine) at the polymorphic site, thealtered allele can contain a “C” (cytidine), “G” (guanine), or “A”(adenine) at the polymorphic site.

SNP's may occur in protein-coding nucleic acid sequences, in which casethey may give rise to a defective or otherwise variant protein, orgenetic disease. Such a SNP may alter the coding sequence of the geneand therefore specify another amino acid (a “missense” SNP) or a SNP mayintroduce a stop codon (a “nonsense” SNP). When a SNP does not alter theamino acid sequence of a protein, the SNP is called “silent.” SNP's mayalso occur in noncoding regions of the nucleotide sequence. This mayresult in defective protein expression, e.g., as a result of alternativespicing, or it may have no effect.

The term “linkage” describes the tendency of genes, alleles, loci orgenetic markers to be inherited together as a result of their locationon the same chromosome. It can be measured by percent recombinationbetween the two genes, alleles, loci, or genetic markers. The term“linkage disequilibrium,” also referred to herein as “LD,” refers to agreater than random association between specific alleles at two markerloci within a particular population. In general, linkage disequilibriumdecreases with an increase in physical distance. If linkagedisequilibrium exists between two markers, or SNPs, then the genotypicinformation at one marker, or SNP, can be used to make probabilisticpredictions about the genotype of the second marker.

As used herein, a “transgenic animal” is a non-human animal, preferablya mammal, more preferably a rodent such as a rat or mouse, in which oneor more of the cells of the animal includes a transgene. Other examplesof transgenic animals include non-human primates, sheep, dogs, cows,goats, chickens, amphibians, etc. A transgene is exogenous DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops and which remains in the genome of the mature animal, therebydirecting the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal. The transgene is introducedinto the cell, directly or indirectly by introduction into a precursorof the cell, e.g., by microinjection, transfection or infection, e.g.,by infection with a recombinant virus. The term genetic manipulationincludes the introduction of a recombinant DNA molecule. This moleculemay be integrated within a chromosome, or it may be extrachromosomallyreplicating DNA.

As used herein, an “homologous recombinant animal” is a non-humananimal, preferably a mammal, more preferably a mouse, in which anendogenous gene has been altered by homologous recombination between theendogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell of the animal, prior to developmentof the animal. Transgenic animals also include inducible transgenicanimals, such as those described in, for example, Chan I. T., et al.(2004) J Clin Invest. 113(4):528-38 and Chin L. et al (1999) Nature400(6743):468-72.

As used herein, the term “rodent” refers to all members of thephylogenetic order Rodentia.

As used herein, the term “misexpression” includes a non-wild typepattern of gene expression. Expression as used herein includestranscriptional, post transcriptional, e.g., mRNA stability,translational, and post translational stages. Misexpression includes:expression at non-wild type levels, i.e., over or under expression; apattern of expression that differs from wild type in terms of the timeor stage at which the gene is expressed, e.g., increased or decreasedexpression (as compared with wild type) at a predetermined developmentalperiod or stage; a pattern of expression that differs from wild type interms of decreased expression (as compared with wild type) in apredetermined cell type or tissue type; a pattern of expression thatdiffers from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.Misexpression includes any expression from a transgenic nucleic acid.Misexpression includes the lack or non-expression of a gene ortransgene, e.g., that can be induced by a deletion of all or part of thegene or its control sequences.

As used herein, the term “knockout” refers to an animal or celltherefrom, in which the insertion of a transgene disrupts an endogenousgene in the animal or cell therefrom. This disruption can essentiallyeliminate PGC-1α in the animal or cell.

In preferred embodiments, misexpression of the gene encoding the PGC-1αprotein is caused by disruption of the PGC-1α gene. For example, thePGC-1α gene can be disrupted through removal of DNA encoding all or partof the protein.

In preferred embodiments, the animal can be heterozygous or homozygousfor a misexpressed PGC-1α gene, e.g., it can be a transgenic animalheterozygous or homozygous for a PGC-1α transgene.

In preferred embodiments, the animal is a transgenic mouse with atransgenic disruption of the PGC-1α gene, preferably an insertion ordeletion, which inactivates the gene product.

In another aspect, the invention features, a nucleic acid moleculewhich, when introduced into an animal or cell, results in themisexpression of the PGC-1α gene in the animal or cell. In preferredembodiments, the nucleic acid molecule includes a PGC-1α nucleotidesequence which includes a disruption, e.g., an insertion or deletion andpreferably the insertion of a marker sequence.

As used herein, “disruption of a gene” refers to a change in the genesequence, e.g., a change in the coding region. Disruption includesinsertions, deletions, point mutations, and rearrangements, e.g.,inversions. The disruption can occur in a region of the native PGC-1αDNA sequence (e.g., one or more exons) and/or the promoter region of thegene so as to decrease or prevent expression of the gene in a cell ascompared to the wild-type or naturally occurring sequence of the gene.The “disruption” can be induced by classical random mutation or by sitedirected methods. Disruptions can be transgenically introduced. Thedeletion of an entire gene is a disruption. Preferred disruptions reducePGC-1α levels to about 50% of wild type, in heterozygotes or essentiallyeliminate PGC-1α in homozygotes.

As used herein, the term “transgenic cell” refers to a cell containing atransgene.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules (organic orinorganic) or other drugs) which bind to PGC-1α proteins, have astimulatory or inhibitory effect on, for example, PGC-1α expression orPGC-1α activity, or have a stimulatory or inhibitory effect on, forexample, the expression or activity of a PGC-1α substrate. Compoundsidentified using assays described herein may be useful for modulatingPGC-1α expression or activity, e.g., increasing PGC-1α expression oractivity. Thus, these compounds would be useful for treating orpreventing neurological diseases or disorders.

These assays are designed to identify compounds that bind to or interactwith a PGC-1α protein, or bind to or interact with other intracellularor extracellular proteins that interact with or modulate a PGC-1αprotein. Such compounds may include, but are not limited to peptides,antibodies, nucleic acid molecules, siRNA molecules, or small organic orinorganic compounds. Such compounds may also include other cellularproteins.

Compounds identified via assays such as those described herein may beuseful, for example, modulating PGC-1α, e.g., by causing increasedPGC-1α expression or activity and, for example, decreased lesionformation, increased neurite growth and decreased mitochondrialdysfunction. Thus, these compounds would be useful for treating orpreventing a neurological disease or disorder. In instances wherebyincreased PGC-1α activity or expression is desired compounds thatinteract with the PGC-1α protein may include compounds which accentuateor amplify the expression or activity of PGC-1α protein. Such compoundswould bring about an effective increase in the level of PGC-1α proteinactivity, thus identifying, treating or preventing neurological diseasesor disorders. For example, a partial agonist or an agonist administeredin a dosage or for a length of time to increase expression or activityof PGC-1α would act to increase mitochondrial function, reduce lesionformation, induce neurite growth, and treat or prevent a neurologicaldisease or disorder. Alternatively, in instances whereby decreasedPGC-1α activity or expression is desired, e.g., to induce symptoms of aneurological disease or disorder in an animal model or to induce weightloss, compounds that interact with the PGC-1α protein may includecompounds which inhibit or suppress the expression or activity of PGC-1αprotein. Such compounds would bring about an effective decrease in thelevel of PGC-1α protein activity, thus acting as an inducer for aneurological disease or disorder, depending on the dosage of thecompound and the length of time the compound is administered.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of or interact with a PGC-1αprotein or polypeptide or biologically active portion thereof. Inanother embodiment, the invention provides assays for screeningcandidate or test compounds which bind to or modulate the activity of aPGC-1α protein or polypeptide or biologically active portion thereof.The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. (1997) Anticancer DrugDes. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner USP '409), plasmids (Cull etal. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott andSmith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406);(Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici(1991) J. Mol. Biol. 222:301-310); (Ladner supra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a PGC-1α protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate PGC-1α activity is determined. Determining the ability of thetest compound to modulate PGC-1α activity can be accomplished bymonitoring, for example, intracellular calcium, IP₃, cAMP, ordiacylglycerol concentration, or the phosphorylation profile ofintracellular proteins, or the level of transcription of downstreamgenes. The cell can be of mammalian origin, e.g., a neuron. In oneembodiment, compounds that interact with PGC-1α binding site can bescreened for their ability to function as ligands, i.e., to bind toPGC-1α binding site and modulate transcription or modulate a signaltransduction pathway. Identification of PGC-1α ligands, and measuringthe activity of the ligand-PGC-1α complex, leads to the identificationof modulators (e.g., antagonists or agonists) of this interaction. Suchmodulators may be useful in the treatment and prevention of aneurological disease or disorder modulation of PGC-1α, e.g., by causingincreased expression or activity of PGC-1α.

The ability of the test compound to modulate PGC-1α binding to asubstrate or to bind to PGC-1α can also be determined. Determining theability of the test compound to modulate PGC-1α binding to a substratecan be accomplished, for example, by coupling the PGC-1α substrate witha radioisotope or enzymatic label such that binding of the PGC-1αsubstrate to PGC-1α can be determined by detecting the labeled PGC-1αsubstrate in a complex. PGC-1α could also be coupled with a radioisotopeor enzymatic label to monitor the ability of a test compound to modulatePGC-1α binding to a PGC-1α substrate in a complex. Determining theability of the test compound to bind PGC-1α can be accomplished, forexample, by coupling the compound with a radioisotope or enzymatic labelsuch that binding of the compound to PGC-1α can be determined bydetecting the labeled PGC-1α compound in a complex. For example,compounds (e.g., PGC-1α ligands or substrates) can be labeled with ¹²⁵I,³⁵S, 14C, or ³H, either directly or indirectly, and the radioisotopedetected by direct counting of radioemmission or by scintillationcounting. Compounds can further be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and the enzymatic label detected by determination of conversion of anappropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound (e.g., a PGC-1α ligand or substrate) to interact withPGC-1α without the labeling of any of the interactants. For example, amicrophysiometer can be used to detect the interaction of a compoundwith PGC-1α without the labeling of either the compound or the PGC-1α(McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a“microphysiometer” (e.g., Cytosensor) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometric sensor (LAPS). Changes in thisacidification rate can be used as an indicator of the interactionbetween a compound and PGC-1α.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a PGC-1α target molecule (e.g., a PGC-1αsubstrate) with a test compound and determining the ability of the testcompound to modulate (e.g., stimulate or inhibit) the activity of thePGC-1α target molecule. Determining the ability of the test compound tomodulate the activity of a PGC-1α target molecule can be accomplished,for example, by determining the ability of the PGC-1α protein to bind toor interact with the PGC-1α target molecule.

Determining the ability of the PGC-1α protein or a biologically activefragment thereof, to bind to or interact with a PGC-1α target moleculecan be accomplished by one of the methods described above fordetermining direct binding. In a preferred embodiment, determining theability of the PGC-1α protein to bind to or interact with a PGC-1αtarget molecule can be accomplished by determining the activity of thetarget molecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular second messenger of thetarget (i.e., intracellular Ca²⁺, diacylglycerol, IP₃, cAMP), detectingcatalytic/enzymatic activity of the target on an appropriate substrate,detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., luciferase), or detecting atarget-regulated cellular response (e.g., gene expression).

In yet another embodiment, an assay of the present invention is acell-free assay in which a PGC-1α protein or biologically active portionthereof, is contacted with a test compound and the ability of the testcompound to bind to the PGC-1α protein or biologically active portionthereof is determined. Preferred biologically active portions of thePGC-1α proteins to be used in assays of the present invention includefragments which participate in interactions with non-PGC-1α molecules,e.g., fragments with high surface probability scores. Binding of thetest compound to the PGC-1α protein can be determined either directly orindirectly as described above. In a preferred embodiment, the assayincludes contacting the PGC-1α protein or biologically active portionthereof with a known compound which binds PGC-1α to form an assaymixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with a PGC-1αprotein, wherein determining the ability of the test compound tointeract with a PGC-1α protein comprises determining the ability of thetest compound to preferentially bind to PGC-1α or biologically activeportion thereof as compared to the known compound. Compounds thatmodulate the interaction of PGC-1α with a known target protein may beuseful in regulating the activity of a PGC-1α protein, especially amutant PGC-1α protein.

In another embodiment, the assay is a cell-free assay in which a PGC-1αprotein or biologically active portion thereof is contacted with a testcompound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the PGC-1α protein or biologicallyactive portion thereof is determined. Determining the ability of thetest compound to modulate the activity of a PGC-1α protein can beaccomplished, for example, by determining the ability of the PGC-1αprotein to bind to a PGC-1α target molecule by one of the methodsdescribed above for determining direct binding. Determining the abilityof the PGC-1α protein to bind to a PGC-1α target molecule can also beaccomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA) (Sjolander, S, and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705). As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In another embodiment, determining the ability of the test compound tomodulate the activity of a PGC-1α protein can be accomplished bydetermining the ability of the PGC-1α protein to further modulate theactivity of a downstream effector of a PGC-1α target molecule. Forexample, the activity of the effector molecule on an appropriate targetcan be determined or the binding of the effector to an appropriatetarget can be determined as previously described.

In yet another embodiment, the cell-free assay involves contacting aPGC-1α protein or biologically active portion thereof with a knowncompound which binds the PGC-1α protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the PGC-1α protein,wherein determining the ability of the test compound to interact withthe PGC-1α protein comprises determining the ability of the PGC-1αprotein to preferentially bind to or modulate the activity of a PGC-1αtarget molecule.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either PGC-1α or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to a PGC-1α protein, or interaction ofa PGC-1α protein with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/PGC-1α fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or PGC-1α protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofPGC-1α binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a PGC-1αprotein or a PGC-1α target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated PGC-1α protein ortarget molecules can be prepared from biotin-NHS(N-hydroxy-succinimide)using techniques known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with PGC-1α protein or target molecules but which donot interfere with binding of the PGC-1α protein to its target moleculecan be derivatized to the wells of the plate, and unbound target orPGC-1α protein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the PGC-1α protein or target molecule, as wellas enzyme-linked assays which rely on detecting an enzymatic activityassociated with the PGC-1α protein or target molecule.

In another embodiment, modulators of PGC-1α expression are identified ina method wherein a cell is contacted with a candidate compound and theexpression of PGC-1α mRNA or protein in the cell is determined. Thelevel of expression of PGC-1α mRNA or protein in the presence of thecandidate compound is compared to the level of expression of PGC-1α mRNAor protein in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of PGC-1α expressionbased on this comparison. For example, when expression of PGC-1α mRNA orprotein is greater (statistically significantly greater) in the presenceof the candidate compound than in its absence, the candidate compound isidentified as a stimulator of PGC-1α mRNA or protein expression.Alternatively, when expression of PGC-1α mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of PGC-1α mRNA or protein expression. The level of PGC-1α mRNAor protein expression in the cells can be determined by methodsdescribed herein for detecting PGC-1α mRNA or protein.

In yet another aspect of the invention, the PGC-1α proteins can be usedas “bait proteins” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with PGC-1α (“PGC-1α-binding proteins” or“PGC-1α-bp”) and are involved in PGC-1α activity. Such PGC-1α-bindingproteins are also likely to be involved in the propagation of signals bythe PGC-1α proteins or PGC-1α targets as, for example, downstreamelements of a PGC-1α-mediated signaling pathway. Alternatively, suchPGC-1α-binding proteins are likely to be PGC-1α inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a PGC-1α proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aPGC-1α-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the PGC-1α protein.

In a further embodiment, assays may be devised through the use of theinvention for the purpose of identifying compounds which modulate (e.g.,affect either positively or negatively) interactions between PGC-1α andits substrates and/or binding partners. Such compounds can include, butare not limited to, molecules such as small molecules, antibodies,peptides, hormones, oligonucleotides, nucleic acids, and analogsthereof. Such compounds may also be obtained from any available source,including systematic libraries of natural and/or synthetic compounds.The preferred assay components for use in this embodiment is atranscriptional coactivator, PGC-1α identified herein, the known bindingpartner and/or substrate of same, and the test compound. Test compoundscan be supplied from any source.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between PGC-1α and its binding partnerinvolves preparing a reaction mixture containing PGC-1α and its bindingpartner under conditions and for a time sufficient to allow the twoproducts to interact and bind, thus forming a complex. In order to testan agent for inhibitory activity, the reaction mixture is prepared inthe presence and absence of the test compound. The test compound can beinitially included in the reaction mixture, or can be added at a timesubsequent to the addition of PGC-1α and its binding partner. Controlreaction mixtures are incubated without the test compound or with aplacebo. The formation of any complexes between PGC-1α and its bindingpartner is then detected. The formation of a complex in the controlreaction, but less or no such formation in the reaction mixturecontaining the test compound, indicates that the compound interfereswith the interaction of PGC-1α and its binding partner. Conversely, theformation of more complex in the presence of compound than in thecontrol reaction indicates that the compound may enhance interaction ofPGC-1α and its binding partner.

The assay for compounds that interfere with the interaction of PGC-1αwith its binding partner may be conducted in a heterogeneous orhomogeneous format. Heterogeneous assays involve anchoring either PGC-1αor its binding partner onto a solid phase and detecting complexesanchored to the solid phase at the end of the reaction. In homogeneousassays, the entire reaction is carried out in a liquid phase. In eitherapproach, the order of addition of reactants can be varied to obtaindifferent information about the compounds being tested. For example,test compounds that interfere with the interaction between PGC-1α andthe binding partners (e.g., by competition) can be identified byconducting the reaction in the presence of the test substance, i.e., byadding the test substance to the reaction mixture prior to orsimultaneously with PGC-1α and its interactive binding partner.Alternatively, test compounds that disrupt preformed complexes, e.g.,compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are briefly described below.

In a heterogeneous assay system, either PGC-1α or its binding partner isanchored onto a solid surface or matrix, while the other correspondingnon-anchored component may be labeled, either directly or indirectly. Inpractice, microtitre plates are often utilized for this approach. Theanchored species can be immobilized by a number of methods, eithernon-covalent or covalent, that are typically well known to one whopractices the art. Non-covalent attachment can often be accomplishedsimply by coating the solid surface with a solution of PGC-1α or itsbinding partner and drying. Alternatively, an immobilized antibodyspecific for the assay component to be anchored can be used for thispurpose. Such surfaces can often be prepared in advance and stored.

In related embodiments, a fusion protein can be provided which adds adomain that allows one or both of the assay components to be anchored toa matrix. For example, glutathione-S-transferase/PGC-1α fusion proteinsor glutathione-S-transferase/binding partner can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedPGC-1α or its binding partner, and the mixture incubated underconditions conducive to complex formation (e.g., physiologicalconditions). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound assay components; the immobilizedcomplex assessed either directly or indirectly, for example, asdescribed above. Alternatively, the complexes can be dissociated fromthe matrix, and the level of PGC-1α binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either PGC-1α orPGC-1α binding partner can be immobilized utilizing conjugation ofbiotin and streptavidin. Biotinylated PGC-1α protein or target moleculescan be prepared from biotin-NHS(N-hydroxy-succinimide) using techniquesknown in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). In certain embodiments, theprotein-immobilized surfaces can be prepared in advance and stored.

In order to conduct the assay, the corresponding partner of theimmobilized assay component is exposed to the coated surface with orwithout the test compound. After the reaction is complete, unreactedassay components are removed (e.g., by washing) and any complexes formedwill remain immobilized on the solid surface. The detection of complexesanchored on the solid surface can be accomplished in a number of ways.Where the non-immobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the initially non-immobilizedspecies (the antibody, in turn, can be directly labeled or indirectlylabeled with, e.g., a labeled anti-Ig antibody). Depending upon theorder of addition of reaction components, test compounds which modulate(inhibit or enhance) complex formation or which disrupt preformedcomplexes can be detected.

In an alternate embodiment of the invention, a homogeneous assay may beused. This is typically a reaction, analogous to those mentioned above,which is conducted in a liquid phase in the presence or absence of thetest compound. The formed complexes are then separated from unreactedcomponents, and the amount of complex formed is determined. As mentionedfor heterogeneous assay systems, the order of addition of reactants tothe liquid phase can yield information about which test compoundsmodulate (inhibit or enhance) complex formation and which disruptpreformed complexes.

In such a homogeneous assay, the reaction products may be separated fromunreacted assay components by any of a number of standard techniques,including but not limited to: differential centrifugation,chromatography, electrophoresis and immunoprecipitation. In differentialcentrifugation, complexes of molecules may be separated from uncomplexedmolecules through a series of centrifugal steps, due to the differentsedimentation equilibria of complexes based on their different sizes anddensities (see, for example, Rivas, G., and Minton, A. P., TrendsBiochem Sci 1993 August; 18(8):284-7). Standard chromatographictechniques may also be utilized to separate complexed molecules fromuncomplexed ones. For example, gel filtration chromatography separatesmolecules based on size, and through the utilization of an appropriategel filtration resin in a column format; for example, the relativelylarger complex may be separated from the relatively smaller uncomplexedcomponents. Similarly, the relatively different charge properties of thecomplex as compared to the uncomplexed molecules may be exploited todifferentially separate the complex from the remaining individualreactants, for example through the use of ion-exchange chromatographyresins. Such resins and chromatographic techniques are well known to oneskilled in the art (see, e.g., Heegaard, 1998, J Mol. Recognit.11:141-148; Hage and Tweed, 1997, J. Chromatogr. B. Biomed. Sci. Appl.,699:499-525). Gel electrophoresis may also be employed to separatecomplexed molecules from unbound species (see, e.g., Ausubel et al(eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, NewYork. 1999). In this technique, protein or nucleic acid complexes areseparated based on size or charge, for example. In order to maintain thebinding interaction during the electrophoretic process, nondenaturinggels in the absence of reducing agent are typically preferred, butconditions appropriate to the particular interactants will be well knownto one skilled in the art. Immunoprecipitation is another commontechnique utilized for the isolation of a protein-protein complex fromsolution (see, e.g., Ausubel et al (eds.), In: Current Protocols inMolecular Biology, J. Wiley & Sons, New York. 1999). In this technique,all proteins binding to an antibody specific to one of the bindingmolecules are precipitated from solution by conjugating the antibody toa polymer bead that may be readily collected by centrifugation. Thebound assay components are released from the beads (through a specificproteolysis event or other technique well known in the art which willnot disturb the protein-protein interaction in the complex), and asecond immunoprecipitation step is performed, this time utilizingantibodies specific for the correspondingly different interacting assaycomponent. In this manner, only formed complexes should remain attachedto the beads. Variations in complex formation in both the presence andthe absence of a test compound can be compared, thus offeringinformation about the ability of the compound to modulate interactionsbetween PGC-1α and its binding partner.

Also within the scope of the present invention are methods for directdetection of interactions between PGC-1α and its natural binding partnerand/or a test compound in a homogeneous or heterogeneous assay systemwithout further sample manipulation. For example, the technique offluorescence energy transfer may be utilized (see, e.g., Lakowicz et al,U.S. Pat. No. 5,631,169; Stavrianopoulos et al, U.S. Pat. No.4,868,103). Generally, this technique involves the addition of afluorophore label on a first ‘donor’molecule (e.g., PGC-1α or testcompound) such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule (e.g., PGC-1α or testcompound), which in turn is able to fluoresce due to the absorbedenergy. Alternately, the ‘donor’ protein molecule may simply utilize thenatural fluorescent energy of tryptophan residues. Labels are chosenthat emit different wavelengths of light, such that the ‘acceptor’molecule label may be differentiated from that of the ‘donor’. Since theefficiency of energy transfer between the labels is related to thedistance separating the molecules, spatial relationships between themolecules can be assessed. In a situation in which binding occursbetween the molecules, the fluorescent emission of the ‘acceptor’molecule label in the assay should be maximal. An FET binding event canbe conveniently measured through standard fluorometric detection meanswell known in the art (e.g., using a fluorimeter). A test substancewhich either enhances or hinders participation of one of the species inthe preformed complex will result in the generation of a signal variantto that of background. In this way, test substances that modulateinteractions between PGC-1α and its binding partner can be identified incontrolled assays.

In another embodiment, modulators of PGC-1α expression are identified ina method wherein a cell is contacted with a candidate compound and theexpression of mRNA or protein, corresponding to a PGC-1α in the cell, isdetermined. The level of expression of mRNA or protein in the presenceof the candidate compound is compared to the level of expression of mRNAor protein in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of PGC-1α expressionbased on this comparison. For example, when expression of PGC-1α mRNA orprotein is greater (statistically significantly greater) in the presenceof the candidate compound than in its absence, the candidate compound isidentified as a stimulator of PGC-1α mRNA or protein expression.Conversely, when expression of PGC-1α mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of PGC-1α mRNA or protein expression. The level of PGC-1α mRNAor protein expression in the cells can be determined by methodsdescribed herein for detecting PGC-1α mRNA or protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a PGC-1α protein can beconfirmed in vivo, e.g., in an animal such as an animal model for aneurological disease or disorder, as described herein, or described in,for example, Sathasivam K et al. Philos Trans R Soc Lond B Biol Sci.1999 Jun. 29; 354(1386):963-9; Bates G P, et al. Hum Mol. Genet. 1997;6(10):1633-7; Shaw C A et al Neurosci Biobehav Rev. 2003 October;27(6):493-505; Menalled LB Trends Pharmacol Sci. 2002 January;23(1):32-9; Legare M E et al. Genet Mol. Res. 2003 Sep. 30; 2(3):288-94;Oiwa Y J Neurosurg. 2003 January; 98(1):136-44; and Bard F et al. Nat.Med. 2000 August; 6(8):916-9, the contents of which are incorporated byreference herein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a small molecule, an antisense PGC-1α nucleicacid molecule, a PGC-1α-specific antibody, or a PGC-1α-binding partner)can be used in an animal model to determine the efficacy, toxicity, orside effects of treatment with such an agent. Alternatively, an agentidentified as described herein can be used in an animal model todetermine the mechanism of action of such an agent. Furthermore, thisinvention pertains to uses of novel agents identified by theabove-described screening assays for treatments as described herein.

Any of the compounds, including but not limited to compounds such asthose identified in the foregoing assay systems, may be tested for acompound capable of treating or preventing a neurological disease ordisorder comprising the ability of the compound to modulate PGC-1αnucleic acid expression or PGC-1α polypeptide activity, therebyidentifying a compound capable of treating or preventing a neurologicaldisease or disorder. Cell-based and animal model-based assays for theidentification of compounds exhibiting such an ability to treat orprevent a neurological disease or disorder described herein.

In one aspect, cell-based systems, as described herein, may be used toidentify compounds which may act to modulate PGC-1α nucleic acidexpression or PGC-1α polypeptide activity or treat neurological diseasesor disorders. For example, such cell systems may be exposed to acompound, suspected of exhibiting an ability to modulate PGC-1α or treator prevent a neurological disease or disorder, at a sufficientconcentration and for a time sufficient to elicit such an ameliorationof disease symptoms in the exposed cells. After exposure, the cells areexamined to determine whether one or more of the disease phenotypes,e.g., Huntington's Disease, for example, has been altered to resemble amore normal or more wild type disease phenotype.

In addition, animal-based disease systems, such as those describedherein, may be used to identify compounds which may act to modulatePGC-1α nucleic acid expression or PGC-1α polypeptide activity or treatneurological diseases or disorders. Such animal models may be used astest substrates for the identification of drugs, pharmaceuticals,therapies, and interventions which may be effective in modulatingPGC-1α, treating or preventing neurological diseases or disorders e.g.,Huntington's disease.

In one embodiment, compounds which are capable of treating or preventinga neurological disease or disorder are identified by assaying theability of the compound to modulate PGC-1α nucleic acid expression orPGC-1α polypeptide activity is determined by detecting modulation ofmitochondrial function, e.g., mitochondrial function in the brain.Furthermore, the invention includes identifying compound which have theability to modulate PGC-1α nucleic acid expression or PGC-1α polypeptideactivity is determined by detecting modulation in the expression oractivity of mitochondrial genes, e.g., LDH2, Ndufb5, COX6a1, and ATP5j.

In still another embodiment, compounds which are capable of treating orpreventing a neurological disease or disorder are identified by assayingthe ability of the compound to modulate PGC-1α is determined bydetecting modulation in the expression or activity of neuronal genes,e.g., NF—H, NF-M, MOBP, ATPa1, and ATP1a2.

Additionally, gene expression patterns may be utilized to assess theability of a compound to modulate PGC-1 e.g., by causing increasedPGC-1α expression or activity. Thus, these compounds would be useful fortreating, preventing, or assessing a neurological disease or disorder.For example, the expression pattern of one or more genes may form partof a “gene expression profile” or “transcriptional profile” which may bethen be used in such an assessment. “Gene expression profile” or“transcriptional profile”, as used herein, includes the pattern of mRNAexpression obtained for a given tissue or cell type under a given set ofconditions. Gene expression profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR. In one embodiment, PGC-1α gene sequences may be used as probesand/or PCR primers for the generation and corroboration of such geneexpression profiles.

Gene expression profiles may be characterized for known states withinthe cell- and/or animal-based model systems. Subsequently, these knowngene expression profiles may be compared to ascertain the effect a testcompound has to modify such gene expression profiles, and to cause theprofile to more closely resemble that of a more desirable profile.

II. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating or preventing a neurological disease or disorder ina subject, e.g., a human, at risk of (or susceptible to) a neurologicaldisease or disorder, by administering to said subject a PGC-1αmodulator, such that the neurological disease or disorder is treated orprevented. In a preferred embodiment, which includes both prophylacticand therapeutic methods, the PGC-1α modulator is administered by in apharmaceutically acceptable formulation.

With regard to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics,” as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers to the study of how apatient's genes determine his or her response to a drug (e.g., apatient's “drug response phenotype”, or “drug response genotype”).

Thus, another aspect of the invention provides methods for tailoring asubject's prophylactic or therapeutic treatment with either the PGC-1αmolecules of the present invention or PGC-1α modulators according tothat individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

A. Prophylactic Methods

In one aspect, the invention provides a method for treating orpreventing a neurological disease or disorder by administering to asubject an agent which modulates PGC-1α expression or PGC-1α activity.The invention also provides methods for modulating the formation ofbrain lesions, neurodegneration, and neurite growth in a subject.Subjects at risk for a neurological disease or disorder can beidentified by, for example, any or a combination of the diagnostic orprognostic assays described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofa neurological disease or disorder, such that the neurological diseaseor disorder or symptom thereof, e.g., hyperactivity, is prevented or,alternatively, delayed in its progression. Depending on the type ofPGC-1α aberrancy, for example, a PGC-1α agonist or PGC-1α antagonistagent can be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

B. Therapeutic Methods

The present invention provides methods for modulating PGC-1α in asubject by administering a PGC-1α modulator to either induce or inhibitPGC-1α expression or activity. In one embodiment, PGC-1α expression oractivity is increased by administering an inducer or agonist of PGC-1αexpression or activity, thereby modulating lesion formation, e.g., brainlesion formation, mitochondrial function, neurite growth, and/orneurodegeneration, and treating or preventing a neurological disease ordisorder.

Accordingly, another aspect of the invention pertains to methods ofmodulating PGC-1α expression or activity for therapeutic purposes andfor use in treatment of neurological diseases or disorders. In anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a PGC-1α or agent that modulates one or more ofthe activities of PGC-1α protein activity associated with a neurologicaldisease or disorder (e.g., modulation of mitochondrial function, brainlesion formation, neurite growth or neuronal degeneration). An agentthat modulates PGC-1α protein activity can be an agent as describedherein, such as a nucleic acid or α protein, an siRNA targeting PGC-1αmRNA, a naturally-occurring target molecule of a PGC-1α protein (e.g., aPGC-1α ligand or substrate), a PGC-1α antibody, a PGC-1α agonist orantagonist, a peptidomimetic of a PGC-1α agonist or antagonist, or othersmall molecule. In one embodiment, the agent stimulates one or morePGC-1α activities. Examples of such stimulatory agents include activePGC-1α protein, a nucleic acid molecule encoding PGC-1α, or a smallmolecule agonist, or mimetic, e.g., a peptidomimetic. In anotherembodiment, the agent inhibits one or more PGC-1α activities. Examplesof such inhibitory agents include antisense PGC-1α nucleic acidmolecules, siRNA molecules, anti-PGC-1α antibodies, small molecules, andPGC-1α inhibitors. These modulatory methods can be performed in vitro(e.g., by culturing the cell with the agent) or, alternatively, in vivo(e.g., by administering the agent to a subject). In one embodiment, themethod involves administering an agent (e.g., an agent identified by ascreening assay described herein), or combination of agents thatmodulates (e.g., upregulates or downregulates) PGC-1α expression oractivity. In another embodiment, the method involves administering aPGC-1α protein or nucleic acid molecule as therapy to compensate forreduced, aberrant, or unwanted PGC-1α expression or activity.

Stimulation of PGC-1α activity is desirable in situations in whichPGC-1α is abnormally downregulated and/or in which increased PGC-1αactivity is likely to have a beneficial effect, e.g., as a treatment fora neurological disease or disorder. Likewise, inhibition of PGC-1αactivity is desirable in situations in which PGC-1α is abnormallyupregulated and/or in which decreased PGC-1α activity is likely to havea beneficial effect, e.g., to effect the creation of an animal model fora neurological disease or disorder, e.g., a non-human animal transgenicin which PGC-1α is misexpressed, or to modulate body weight in asubject, e.g., to treat or prevent obesity.

(i) Methods for Increasing PGC-1α Expression or Activity

Increasing PGC-1α expression or activity leads to treatment orprevention of a neurological disease or disorder, therefore providing amethod for treating, preventing, and assessing a neurological disease ordisorder. A variety of techniques may be used to increase theexpression, synthesis, or activity of PGC-1α.

Described in this section are methods whereby the level PGC-1α activitymay be increased, for example, by either increasing the level of PGC-1αgene expression or by increasing the level of active PGC-1α proteinwhich is present.

For example, a PGC-1α protein may be administered to a subject. Any ofthe techniques discussed below may be used for such administration. Oneof skill in the art will readily know how to determine the concentrationof effective, non-toxic doses of the PGC-1α protein, utilizingtechniques such as those described below.

Additionally, RNA sequences encoding a PGC-1α protein may be directlyadministered to a subject, at a concentration sufficient to produce alevel of PGC-1α protein such that PGC-1α is modulated. Any of thetechniques discussed below, which achieve intracellular administrationof compounds, such as, for example, liposome administration, may be usedfor the administration of such RNA molecules. The RNA molecules may beproduced, for example, by recombinant techniques such as those describedherein. Other pharmaceutical compositions, medications, or therapeuticsmay be used in combination with the PGC-1α agonists described herein.Further, subjects may be treated by gene replacement therapy, resultingin permanent modulation of PGC-1α. One or more copies of a PGC-1α gene,or a portion thereof, that directs the production of a normal PGC-1αprotein with PGC-1α function, may be inserted into cells using vectorswhich include, but are not limited to adenovirus, adeno-associatedvirus, and retrovirus vectors, in addition to other particles thatintroduce DNA into cells, such as liposomes. Additionally, techniquessuch as those described above may be used for the introduction of PGC-1αgene sequences into human cells. Furthermore, expression or activity oftranscriptional activators which act upon PGC-1α may be increased tothereby increase expression and activity of PGC-1α. Small moleculeswhich induce PGC-1α expression or activity, either directly orindirectly may also be used. In one embodiment, a small moleculefunctions to disrupt a protein-protein interaction between PGC-1α and atarget molecule or ligand, thereby modulating, e.g., increasing ordecreasing the activity of PGC-1α.

Cells, preferably, autologous cells, containing PGC-1α expressing genesequences may then be introduced or reintroduced into the subject. Suchcell replacement techniques may be preferred, for example, when the geneproduct is a secreted, extracellular gene product.

(ii) Methods for Inhibiting PGC-1α Expression, Synthesis, or Activity

As discussed above, inhibition of PGC-1α expression or activity may bedesirable in certain situations, e.g., to create a non-human animaltransgenic in which PGC-1α is misexpressed, or to modulate body weightin a subject, e.g., to treat or prevent obesity. A variety of techniquesmay be used to inhibit the expression, synthesis, or activity of PGC-1αgenes and/or proteins.

For example, compounds such as those identified through assays describedabove, which exhibit inhibitory activity, may be used in accordance withthe invention. Such molecules may include, but are not limited to, smallorganic molecules, siRNA molecules, peptides, antibodies, and the like.

For example, compounds can be administered that compete with endogenousligand for the PGC-1α protein. The resulting reduction in the amount ofligand-bound PGC-1α protein will modulate endothelial cell physiology.Compounds that can be particularly useful for this purpose include, forexample, soluble proteins or peptides, such as peptides comprising oneor more of the extracellular domains, or portions and/or analogsthereof, of the PGC-1α protein, including, for example, soluble fusionproteins such as Ig-tailed fusion proteins. (For a discussion of theproduction of Ig-tailed fusion proteins, see, for example, U.S. Pat. No.5,116,964). Alternatively, compounds, such as ligand analogs orantibodies, that bind to the PGC-1α receptor site, but do not activatethe protein, (e.g., receptor-ligand antagonists) can be effective ininhibiting PGC-1α protein activity.

Further, antisense and ribozyme molecules and siRNA molecules whichinhibit expression of the PGC-1α gene may also be used in accordancewith the invention to inhibit aberrant PGC-1α gene activity. Stillfurther, triple helix molecules may be utilized in inhibiting aberrantPGC-1α gene activity.

The antisense nucleic acid molecules used in the methods of theinvention are typically administered to a subject or generated in situsuch that they hybridize with or bind to cellular mRNA and/or genomicDNA encoding a PGC-1α protein to thereby inhibit expression of theprotein, e.g., by inhibiting transcription and/or translation. Thehybridization can be by conventional nucleotide complementarity to forma stable duplex, or, for example, in the case of an antisense nucleicacid molecule which binds to DNA duplexes, through specific interactionsin the major groove of the double helix. An example of a route ofadministration of antisense nucleic acid molecules of the inventioninclude direct injection at a tissue site. Alternatively, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, for systemic administration,antisense molecules can be modified such that they specifically bind toreceptors or antigens expressed on a selected cell surface, e.g., bylinking the antisense nucleic acid molecules to peptides or antibodieswhich bind to cell surface receptors or antigens. The antisense nucleicacid molecules can also be delivered to cells using the vectorsdescribed herein. To achieve sufficient intracellular concentrations ofthe antisense molecules, vector constructs in which the antisensenucleic acid molecule is placed under the control of a strong pol II orpol III promoter are preferred.

In yet another embodiment, an antisense nucleic acid molecule used inthe methods of the invention is an α-anomeric nucleic acid molecule. Anα-anomeric nucleic acid molecule forms specific double-stranded hybridswith complementary RNA in which, contrary to the usual β-units, thestrands run parallel to each other (Gaultier et al. (1987) NucleicAcids. Res. 15:6625-6641). The antisense nucleic acid molecule can alsocomprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic AcidsRes. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)FEBS Lett. 215:327-330).

In still another embodiment, an antisense nucleic acid used in themethods of the invention is a ribozyme. Ribozymes are catalytic RNAmolecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can beused to catalytically cleave PGC-1α mRNA transcripts thereby to inhibittranslation of PGC-1α mRNA. A ribozyme having specificity for aPGC-1α-encoding nucleic acid can be designed based upon the nucleotidesequence of a PGC-1α cDNA disclosed herein (i.e., SEQ ID NO:1). Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in a PGC-1α-encoding mRNA (see,for example, Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S.Pat. No. 5,116,742). Alternatively, PGC-1α mRNA can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules (see, for example, Bartel, D. and Szostak, J. W. (1993)Science 261:1411-1418).

PGC-1α gene expression can also be inhibited by targeting nucleotidesequences complementary to the regulatory region of the PGC-1α (e.g.,the PGC-1α promoter and/or enhancers) to form triple helical structuresthat prevent transcription of the PGC-1α gene in target cells (see, forexample, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C.et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992)Bioassays 14(12):807-15).

An RNA interfering agent, e.g., an siRNA molecule, which is targeted toPGC-1α, can also be used in order to inhibit expression of PGC-1α, e.g.,through degradation or specific post-transcriptional gene silencing(PTGS) of the messenger RNA (mRNA) of PGC-1α.

Antibodies that are both specific for the PGC-1α protein and interferewith its activity may also be used to modulate or inhibit PGC-1α proteinfunction. Such antibodies may be generated using standard techniquesdescribed herein, against the PGC-1α protein itself or against peptidescorresponding to portions of the protein. Such antibodies include butare not limited to polyclonal, monoclonal, Fab fragments, single chainantibodies, or chimeric antibodies.

In instances where the target gene protein is intracellular and wholeantibodies are used, internalizing antibodies may be preferred.Lipofectin liposomes may be used to deliver the antibody or a fragmentof the Fab region which binds to the target epitope into cells. Wherefragments of the antibody are used, the smallest inhibitory fragmentwhich binds to the target protein's binding domain is preferred. Forexample, peptides having an amino acid sequence corresponding to thedomain of the variable region of the antibody that binds to the targetgene protein may be used. Such peptides may be synthesized chemically orproduced via recombinant DNA technology using methods well known in theart (described in, for example, Creighton (1983), supra; and Sambrook etal. (1989) supra). Single chain neutralizing antibodies which bind tointracellular target gene epitopes may also be administered. Such singlechain antibodies may be administered, for example, by expressingnucleotide sequences encoding single-chain antibodies within the targetcell population by utilizing, for example, techniques such as thosedescribed in Marasco et al. (1993) Proc. Natl. Acad. Sci. USA90:7889-7893).

C. Pharmaceutical Compositions

The methods of the invention involve administering to a subject an agentwhich modulates PGC-1α expression or activity (e.g., an agent identifiedby a screening assay described herein), or a combination of such agents.In another embodiment, the method involves administering to a subject aPGC-1α protein or nucleic acid molecule as therapy to compensate forreduced, aberrant, or unwanted PGC-1α expression or activity.

Stimulation of PGC-1α activity is desirable in situations in whichPGC-1α is abnormally downregulated and/or in which increased PGC-1αactivity is likely to have a beneficial effect, e.g., as a therapeuticor prophylactic.

The agents which modulate PGC-1α activity can be administered to asubject using pharmaceutical compositions suitable for suchadministration. Such compositions typically comprise the agent (e.g.,nucleic acid molecule, protein, or antibody) and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition used in the therapeutic methods of theinvention is formulated to be compatible with its intended route ofadministration. Examples of routes of administration include parenteral,e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the agentthat modulates PGC-1α activity (e.g., a fragment of a PGC-1α protein oran anti-PGC-1α antibody) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying which yieldsa powder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The agents that modulate PGC-1α activity can also be prepared in theform of suppositories (e.g., with conventional suppository bases such ascocoa butter and other glycerides) or retention enemas for rectaldelivery.

In one embodiment, the agents that modulate PGC-1α activity are preparedwith carriers that will protect the compound against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the agent that modulatesPGC-1α activity and the particular therapeutic effect to be achieved,and the limitations inherent in the art of compounding such an agent forthe treatment of subjects.

Toxicity and therapeutic efficacy of such agents can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and can be expressed as the ratio LD50/ED50.Agents which exhibit large therapeutic indices are preferred. Whileagents that exhibit toxic side effects may be used, care should be takento design a delivery system that targets such agents to the site ofaffected tissue in order to minimize potential damage to uninfectedcells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch PGC-1α modulating agents lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anyagent used in the therapeutic methods of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

In a preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds. It is understood that appropriate doses of smallmolecule agents depends upon a number of factors within the ken of theordinarily skilled physician, veterinarian, or researcher. The dose(s)of the small molecule will vary, for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the composition is to beadministered, if applicable, and the effect which the practitionerdesires the small molecule to have upon the nucleic acid or polypeptideof the invention.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram). It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal (e.g., a human) in order to modulateexpression or activity of a PGC-1α molecule, a physician, veterinarian,or researcher may, for example, prescribe a relatively low dose atfirst, subsequently increasing the dose until an appropriate response isobtained. In addition, it is understood that the specific dose level forany particular animal subject will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated, e.g., the intended use of the agonist or antagonize.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, alpha-interferon, beta-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator; orbiological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

The nucleic acid molecules used in the methods of the invention can beinserted into vectors and used as gene therapy vectors. Gene therapyvectors can be delivered to a subject by, for example, intravenousinjection, local administration (see U.S. Pat. No. 5,328,470) or bystereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad.Sci. USA 91:3054-3057). The pharmaceutical preparation of the genetherapy vector can include the gene therapy vector in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery vector can be produced intact from recombinant cells, e.g.,retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gene delivery system.

III. Predictive Medicine:

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining PGC-1α proteinand/or nucleic acid expression as well as PGC-1α activity, in thecontext of a biological sample (e.g., blood, serum, fluid, e.g.,cerebrospinal fluid, spinal fluid, cells, or tissue, e.g., neuraltissue) to thereby determine whether an individual is afflicted withneurological disease or disorder neurological disease or disorder has arisk of developing a neurological disease or disorder. The inventionalso provides for prognostic (or predictive) assays for determiningwhether an individual is at risk of developing a neurological disease ordisorder. For example, mutations in a PGC-1α gene can be assayed for ina biological sample. Such assays can be used for prognostic orpredictive purpose to thereby phophylactically treat an individual priorto the onset of a neurological disease or disorder.

One particular embodiment includes a method for assessing whether asubject is afflicted with a neurological disease or disorder has a riskof developing a neurological disease or disorder comprising detectingthe expression of the PGC-1α gene or the activity of PGC-1α in a cell ortissue sample of a subject, wherein a decrease in the expression of thePGC-1α gene or a decrease in the activity of PGC-1α indicates thepresence of a neurological disease or disorder or the risk of developinga neurological disease or disorder in the subject. In this embodiment,subject samples tested are, for example, cerebrospinal fluid, spinalfluid, and neural tissue.

Another aspect of the invention pertains to monitoring the influence ofPGC-1α modulators on the expression or activity of PGC-1α in clinicaltrials.

These and other agents are described in further detail in the followingsections.

A. Prognostic and Diagnostic Assays

To determine whether a subject is afflicted with a neurological diseaseor disorder has a risk of developing a neurological disease or disorder,a biological sample may be obtained from a subject and the biologicalsample may be contacted with a compound or an agent capable of detectinga PGC-1α protein or nucleic acid (e.g., mRNA or genomic DNA) thatencodes a PGC-1α protein, in the biological sample. A preferred agentfor detecting PGC-1α mRNA or genomic DNA is a labeled nucleic acid probecapable of hybridizing to PGC-1α mRNA or genomic DNA. The nucleic acidprobe can be, for example, the PGC-1α nucleic acid set forth in SEQ IDNO:1, or a portion thereof, such as an oligonucleotide of at least 15,20, 25, 30, 25, 40, 45, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions toPGC-1α mRNA or genomic DNA. Other suitable probes for use in thediagnostic assays of the invention are described herein.

The term “biological sample” is intended to include tissues, cells, andbiological fluids isolated from a subject, as well as tissues, cells,and fluids present within a subject, e.g., cerebrospinal fluid, spinalfluid, and neural tissue. That is, the detection method of the inventioncan be used to detect PGC-1α mRNA, protein, or genomic DNA in abiological sample in vitro as well as in vivo. For example, in vitrotechniques for detection of PGC-1α mRNA include Northern hybridizationsand in situ hybridizations. In vitro techniques for detection of PGC-1αprotein include enzyme linked immunosorbent assays (ELISAs), Westernblots, immunoprecipitations and immunofluorescence. In vitro techniquesfor detection of PGC-1α genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of PGC-1α protein includeintroducing into a subject a labeled anti-PGC-1α antibody. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting PGC-1α protein, mRNA, orgenomic DNA, such that the presence of PGC-1α protein, mRNA or genomicDNA is detected in the biological sample, and comparing the presence ofPGC-1α protein, mRNA or genomic DNA in the control sample with thepresence of PGC-1α protein, mRNA or genomic DNA in the test sample.

Analysis of one or more PGC-1α polymorphic regions in a subject can beuseful for predicting whether a subject has or is likely to develop aneurological disease or disorder. In preferred embodiments, the methodsof the invention can be characterized as comprising detecting, in asample of cells from the subject, the presence or absence of a specificallelic variant of one or more polymorphic regions of a PGC-1α gene. Theallelic differences can be: (i) a difference in the identity of at leastone nucleotide or (ii) a difference in the number of nucleotides, whichdifference can be a single nucleotide or several nucleotides. Theinvention also provides methods for detecting differences in an PGC-1αgene such as chromosomal rearrangements, e.g., chromosomal dislocation.The invention can also be used in prenatal diagnostics.

A preferred detection method is allele specific hybridization usingprobes overlapping the polymorphic site and having about 5, 10, 20, 25,or 30 nucleotides around the polymorphic region. In a preferredembodiment of the invention, several probes capable of hybridizingspecifically to allelic variants are attached to a solid phase support,e.g., a “chip”. Oligonucleotides can be bound to a solid support by avariety of processes, including lithography. For example, a chip canhold up to 250,000 oligonucleotides (GeneChip, Affymetrix). Mutationdetection analysis using these chips comprising oligonucleotides, alsotermed “DNA probe arrays” is described e.g., in Cronin et al. (1996)Human Mutation 7:244. In one embodiment, a chip comprises all theallelic variants of at least one polymorphic region of a gene. The solidphase support is then contacted with a test nucleic acid andhybridization to the specific probes is detected. Accordingly, theidentity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment. For example, theidentity of the allelic variant of the nucleotide polymorphism in the 5′upstream regulatory element can be determined in a single hybridizationexperiment.

In other detection methods, it is necessary to first amplify at least aportion of a PGC-1α gene prior to identifying the allelic variant.Amplification can be performed, e.g., by PCR and/or LCR (see Wu andWallace, (1989) Genomics 4:560), according to methods known in the art.In one embodiment, genomic DNA of a cell is exposed to two PCR primersand amplification for a number of cycles sufficient to produce therequired amount of amplified DNA. In preferred embodiments, the primersare located between 150 and 350 base pairs apart.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), and self-sustainedsequence replication (Guatelli et al., (1989) Proc. Nat. Acad. Sci.87:1874), and nucleic acid based sequence amplification (NABSA), or anyother nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill inthe art. These detection schemes are especially useful for the detectionof nucleic acid molecules if such molecules are present in very lownumbers.

In one embodiment, any of a variety of sequencing reactions known in theart can be used to directly sequence at least a portion of a PGC-1α geneand detect allelic variants, e.g., mutations, by comparing the sequenceof the sample sequence with the corresponding reference (control)sequence. Exemplary sequencing reactions include those based ontechniques developed by Maxam and Gilbert (Proc. Natl. Acad Sci USA(1977) 74:560) or Sanger (Sanger et al. (1977) Proc. Nat. Acad. Sci.74:5463). It is also contemplated that any of a variety of automatedsequencing procedures may be utilized when performing the subject assays(Biotechniques (1995) 19:448), including sequencing by mass spectrometry(see, for example, U.S. Pat. No. 5,547,835 and international patentapplication Publication Number WO 94/16101, entitled DNA Sequencing byMass Spectrometry by H. Köster; U.S. Pat. No. 5,547,835 andinternational patent application Publication Number WO 94/21822 entitled“DNA Sequencing by Mass Spectrometry Via Exonuclease Degradation” by H.Köster), and U.S. Pat. No. 5,605,798 and International PatentApplication No. PCT/US96/03651 entitled DNA Diagnostics Based on MassSpectrometry by H. Köster; Cohen et al. (1996) Adv Chromatogr36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol38:147-159). It will be evident to one skilled in the art that, forcertain embodiments, the occurrence of only one, two or three of thenucleic acid bases need be determined in the sequencing reaction. Forinstance, A-track or the like, e.g., where only one nucleotide isdetected, can be carried out.

Yet other sequencing methods are disclosed, e.g., in U.S. Pat. No.5,580,732 entitled “Method of DNA sequencing employing a mixedDNA-polymer chain probe” and U.S. Pat. No. 5,571,676 entitled “Methodfor mismatch-directed in vitro DNA sequencing”.

In some cases, the presence of a specific allele of a PGC-1α gene in DNAfrom a subject can be shown by restriction enzyme analysis. For example,a specific nucleotide polymorphism can result in a nucleotide sequencecomprising a restriction site which is absent from the nucleotidesequence of another allelic variant.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, thetechnique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing a control nucleic acid, which is optionallylabeled, e.g., RNA or DNA, comprising a nucleotide sequence of an PGC-1αallelic variant with a sample nucleic acid, e.g., RNA or DNA, obtainedfrom a tissue sample. The double-stranded duplexes are treated with anagent which cleaves single-stranded regions of the duplex such asduplexes formed based on basepair mismatches between the control andsample strands. For instance, RNA/DNA duplexes can be treated with RNaseand DNA/DNA hybrids treated with S1 nuclease to enzymatically digest themismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing polyacrylamide gels to determine whether the control andsample nucleic acids have an identical nucleotide sequence or in whichnucleotides they are different. See, for example, Cotton et al. (1988)Proc. Natl Acad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control or sample nucleicacid is labeled for detection.

In another embodiment, an allelic variant can be identified bydenaturing high-performance liquid chromatography (DHPLC) (Oefner andUnderhill, (1995) Am. J. Human Gen. 57:Suppl. A266). DHPLC usesreverse-phase ion-pairing chromatography to detect the heteroduplexesthat are generated during amplification of PCR fragments fromindividuals who are heterozygous at a particular nucleotide locus withinthat fragment (Oefner and Underhill (1995) Am. J. Human Gen. 57:Suppl.A266). In general, PCR products are produced using PCR primers flankingthe DNA of interest. DHPLC analysis is carried out and the resultingchromatograms are analyzed to identify base pair alterations ordeletions based on specific chromatographic profiles (see O'Donovan etal. (1998) Genomics 52:44-49).

In other embodiments, alterations in electrophoretic mobility is used toidentify the type of PGC-1α allelic variant. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci. USA 86:2766; see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control nucleicacids are denatured and allowed to renature. The secondary structure ofsingle-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In anotherpreferred embodiment, the subject method utilizes heteroduplex analysisto separate double stranded heteroduplex molecules on the basis ofchanges in electrophoretic mobility (Keen et al. (1991) Trends Genet.7:5).

In yet another embodiment, the identity of an allelic variant of apolymorphic region is obtained by analyzing the movement of a nucleicacid comprising the polymorphic region in polyacrylamide gels containinga gradient of denaturant is assayed using denaturing gradient gelelectrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGEis used as the method of analysis, DNA will be modified to insure thatit does not completely denature, for example by adding a GC clamp ofapproximately 40 bp of high-melting GC-rich DNA by PCR. In a furtherembodiment, a temperature gradient is used in place of a denaturingagent gradient to identify differences in the mobility of control andsample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:1275).

Examples of techniques for detecting differences of at least onenucleotide between two nucleic acids include, but are not limited to,selective oligonucleotide hybridization, selective amplification, orselective primer extension. For example, oligonucleotide probes may beprepared in which the known polymorphic nucleotide is placed centrally(allele-specific probes) and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. NatlAcad. Sci. USA 86:6230; and Wallace et al. (1979) Nucl. Acids Res.6:3543). Such allele specific oligonucleotide hybridization techniquesmay be used for the simultaneous detection of several nucleotide changesin different polylmorphic regions of PGC-1α. For example,oligonucleotides having nucleotide sequences of specific allelicvariants are attached to a hybridizing membrane and this membrane isthen hybridized with labeled sample nucleic acid. Analysis of thehybridization signal will then reveal the identity of the nucleotides ofthe sample nucleic acid.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the allelic variant of interest in the center of the molecule(so that amplification depends on differential hybridization) (Gibbs etal. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end ofone primer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238; Newton etal. (1989) Nucl. Acids Res. 17:2503). This technique is also termed“PROBE” for Probe Oligo Base Extension. In addition, it may be desirableto introduce a novel restriction site in the region of the mutation tocreate cleavage-based detection (Gasparini et al. (1992) Mol. Cell.Probes 6:1).

In another embodiment, identification of the allelic variant is carriedout using an oligonucleotide ligation assay (OLA), as described, e.g.,in U.S. Pat. No. 4,998,617 and in Landegren, U. et al., (1988) Science241:1077-1080. The OLA protocol uses two oligonucleotides which aredesigned to be capable of hybridizing to abutting sequences of a singlestrand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al., (1990)Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927. In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA.

Several techniques based on this OLA method have been developed and canbe used to detect specific allelic variants of a polymorphic region ofan PGC-1α gene. For example, U.S. Pat. No. 5,593,826 discloses an OLAusing an oligonucleotide having 3′-amino group and a 5′-phosphorylatedoligonucleotide to form a conjugate having a phosphoramidate linkage. Inanother variation of OLA described in Tobe et al. ((1996) Nucleic AcidsRes 24: 3728), OLA combined with PCR permits typing of two alleles in asingle microtiter well. By marking each of the allele-specific primerswith a unique hapten, i.e. digoxigenin and fluorescein, each OLAreaction can be detected by using hapten specific antibodies that arelabeled with different enzyme reporters, alkaline phosphatase orhorseradish peroxidase. This system permits the detection of the twoalleles using a high throughput format that leads to the production oftwo different colors.

The invention further provides methods for detecting single nucleotidepolymorphisms in a PGC-1α gene. Because single nucleotide polymorphismsconstitute sites of variation flanked by regions of invariant sequence,their analysis requires no more than the determination of the identityof the single nucleotide present at the site of variation and it isunnecessary to determine a complete gene sequence for each subject.Several methods have been developed to facilitate the analysis of suchsingle nucleotide polymorphisms.

In one embodiment, the single base polymorphism can be detected by usinga specialized exonuclease-resistant nucleotide, as disclosed, e.g., inMundy, C. R. (U.S. Pat. No. 4,656,127). According to the method, aprimer complementary to the allelic sequence immediately 3′ to thepolymorphic site is permitted to hybridize to a target molecule obtainedfrom a particular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide presents in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site(Cohen, D. et al. (French Patent 2,650,840; PCT Application No.WO91/02087). As in the Mundy method of U.S. Pat. No. 4,656,127, a primeris employed that is complementary to allelic; sequences immediately 3′to a polymorphic site. The method determines the identity of thenucleotide of that site using labeled dideoxynucleotide derivatives,which, if complementary to the nucleotide of the polymorphic site willbecome incorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA is describedby Goelet, P. et al. (PCT Application No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

Several primer-guided nucleotide incorporation procedures for assayingpolymorphic sites in DNA have been described (Komher, J. S. et al.,Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res.18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1993)). These methods differ from GBA in that they all relyon the incorporation of labeled deoxynucleotides to discriminate betweenbases at a polymorphic site. In such a format, since the signal isproportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A.-C.,et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

For determining the identity of the allelic variant of a polymorphicregion located in the coding region of a PGC-1α gene, yet other methodsthan those described above can be used. For example, identification ofan allelic variant which encodes a mutated PGC-1α protein can beperformed by using an antibody specifically recognizing the mutantprotein in, e.g., immunohistochemistry or immunoprecipitation.Antibodies to wild-type PGC-1α or mutated forms of PGC-1α proteins canbe prepared according to methods known in the art.

Alternatively, one can also measure an activity of a PGC-1α protein,such as binding to a PGC-1α ligand. Binding assays are known in the artand involve, e.g., obtaining cells from a subject, and performingbinding experiments with a labeled lipid, to determine whether bindingto the mutated form of the protein differs from binding to the wild-typeof the protein.

Antibodies directed against reference or mutant PGC-1α polypeptides orallelic variant thereof, which are discussed above, may also be used indisease diagnostics and prognostics. Such diagnostic methods, may beused to detect abnormalities in the level of PGC-1α polypeptideexpression, or abnormalities in the structure and/or tissue, cellular,or subcellular location of an PGC-1α polypeptide. Structural differencesmay include, for example, differences in the size, electronegativity, orantigenicity of the mutant PGC-1α polypeptide relative to the normalPGC-1α polypeptide. Protein from the tissue or cell type to be analyzedmay easily be detected or isolated using techniques which are well knownto one of skill in the art, including but not limited to Western blotanalysis. For a detailed explanation of methods for carrying out Westernblot analysis, see Sambrook et al, 1989, supra, at Chapter 18. Theprotein detection and isolation methods employed herein may also be suchas those described in Harlow and Lane, for example (Harlow, E. and Lane,D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), which is incorporatedherein by reference in its entirety.

This can be accomplished, for example, by immunofluorescence techniquesemploying a fluorescently labeled antibody (see below) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Theantibodies (or fragments thereof) useful in the present invention may,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of PGC-1α polypeptides.In situ detection may be accomplished by removing a histologicalspecimen from a subject, and applying thereto a labeled antibody of thepresent invention. The antibody (or fragment) is preferably applied byoverlaying the labeled antibody (or fragment) onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the PGC-1α polypeptide, but also its distributionin the examined tissue. Using the present invention, one of ordinaryskill will readily perceive that any of a wide variety of histologicalmethods (such as staining procedures) can be modified in order toachieve such in situ detection.

Often a solid phase support or carrier is used as a support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

One means for labeling an anti-PGC-1α polypeptide specific antibody isvia linkage to an enzyme and use in an enzyme immunoassay (EIA) (Voller,“The Enzyme Linked Immunosorbent Assay (ELISA)”, Diagnostic Horizons2:1-7, 1978, Microbiological Associates Quarterly Publication,Walkersville, Md.; Voller, et al., J. Clin. Pathol. 31:507-520 (1978);Butler, Meth. Enzymol. 73:482-523 (1981); Maggio, (ed.) EnzymeImmunoassay, CRC Press, Boca Raton, Fla., 1980; Ishikawa, et al., (eds.)Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Theantibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

If a polymorphic region is located in an exon, either in a coding ornon-coding portion of the gene, the identity of the allelic variant canbe determined by determining the molecular structure of the mRNA,pre-mRNA, or cDNA. The molecular structure can be determined using anyof the above described methods for determining the molecular structureof the genomic DNA.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits, such as those described above, comprisingat least one probe or primer nucleic acid described herein, which may beconveniently used, e.g., to determine whether a subject has or is atrisk of developing a disease associated with a specific PGC-1α allelicvariant.

Sample nucleic acid to be analyzed by any of the above-describeddiagnostic and prognostic methods can be obtained from any cell type ortissue of a subject. For example, a subject's bodily fluid (e.g. blood)can be obtained by known techniques (e.g., venipuncture). Alternatively,nucleic acid tests can be performed on dry samples (e.g., hair or skin).Fetal nucleic acid samples can be obtained from maternal blood asdescribed in International Patent Application No. WO91/07660 to Bianchi.Alternatively, amniocytes or chorionic villi may be obtained forperforming prenatal testing.

Diagnostic procedures may also be performed in situ directly upon tissuesections

(fixed and/or frozen) of subject tissue obtained from biopsies orresections, such that no nucleic acid purification is necessary. Nucleicacid reagents may be used as probes and/or primers for such in situprocedures (see, for example, Nuovo, G. J., 1992, PCR in situhybridization: protocols and applications, Raven Press, NY).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles may also be assessed in such detectionschemes. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

B. Monitoring of Effects During Clinical Trials

The present invention further provides methods for determining theeffectiveness of a PGC-1α modulator (e.g., a PGC-1α modulator identifiedherein) in treating or preventing a neurological disease or disorder orassessing risk of developing a neurological disease or disorder in asubject. For example, the effectiveness of a PGC-1α modulator inincreasing or decreasing PGC-1α gene expression, protein levels, or inupregulating or down-regulating PGC-1α activity, can be monitored inclinical trials of subjects exhibiting increased or decreased PGC-1αgene expression, protein levels, or upregulated or downregulated PGC-1αactivity. In such clinical trials, the expression or activity of aPGC-1α gene, and preferably, other genes that have been implicated in,for example, a PGC-1α pathway can be used as a “read out” or marker ofthe phenotype of a particular cell.

For example, and not by way of limitation, genes, including PGC-1α, thatare modulated in cells by treatment with an agent which modulates PGC-1αactivity (e.g., identified in a screening assay as described herein) canbe identified. Thus, to study the effect of agents which modulate PGC-1αactivity on subjects suffering a neurological disease or disorder, oragents to be used as a prophylactic, for example, a clinical trial,cells can be isolated and RNA prepared and analyzed for the levels ofexpression of PGC-1α and other genes implicated in PGC-1α activity orexpression. The levels of gene expression (e.g., a gene expressionpattern) can be quantified by Northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount of proteinproduced, by one of the methods described herein, or by measuring thelevels of activity of PGC-1α or other genes. In this way, the geneexpression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent which modulates PGC-1αactivity. This response state may be determined before, and at variouspoints during treatment of the individual with the agent which modulatesPGC-1α activity.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agentwhich modulates PGC-1α activity (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, siRNA, or small moleculeidentified by the screening assays described herein) including the stepsof (i) obtaining a pre-administration sample from a subject prior toadministration of the agent; (ii) detecting the level of expression of aPGC-1α protein, mRNA, or genomic DNA in the pre-administration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of thePGC-1α protein, mRNA, or genomic DNA in the post-administration samples;(v) comparing the level of expression or activity of the PGC-1α protein,mRNA, or genomic DNA in the pre-administration sample with the PGC-1αprotein, mRNA, or genomic DNA in the post administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly. For example, increased administration of the agentmay be desirable to increase the expression or activity of PGC-1α tohigher levels than detected, i.e., to increase the effectiveness of theagent. According to such an embodiment, PGC-1α expression or activitymay be used as an indicator of the effectiveness of an agent, even inthe absence of an observable phenotypic response.

IV. Recombinant Expression Vectors and Host Cells Used in the Methods ofthe Invention

The methods of the invention (e.g., the screening assays and therapeuticand/or preventative methods described herein) include the use ofvectors, preferably expression vectors, containing a nucleic acidencoding a PGC-1α protein (or a portion thereof). For example, in oneembodiment, a vector containing a nucleic acid encoding a PGC-1αprotein, or portion thereof, is used to deliver a PGC-1α protein, orportion thereof, to a subject, to treat or prevent a neurologicaldisease or disorder in the subject. In one embodiment, the vectorcontaining a nucleic acid encoding a PGC-1α protein, or portion thereof,is targeted to a specific cell type, organ or tissue, e.g., a brain cellor a specific portion of the brain, e.g., the striatum, using, e.g., atissue specific promoter as described herein.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors to be used in the methods of theinvention comprise a nucleic acid of the invention in a form suitablefor expression of the nucleic acid in a host cell, which means that therecombinant expression vectors include one or more regulatory sequences,selected on the basis of the host cells to be used for expression, whichis operatively linked to the nucleic acid sequence to be expressed.Within a recombinant expression vector, “operably linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequence(s) in a manner which allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel (1990) Methods Enzymol. 185:3-7. Regulatory sequences includethose which direct constitutive expression of a nucleotide sequence inmany types of host cells and those which direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences). It will be appreciated by those skilled in theart that the design of the expression vector can depend on such factorsas the choice of the host cell to be transformed, the level ofexpression of protein desired, and the like. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., PGC-1α proteins, mutant formsof PGC-1α proteins, fusion proteins, and the like).

The recombinant expression vectors to be used in the methods of theinvention can be designed for expression of PGC-1α proteins inprokaryotic or eukaryotic cells. For example, PGC-1α proteins can beexpressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel (1990) supra.Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Purified fusion proteins can be utilized in PGC-1α activity assays,(e.g., direct assays or competitive assays described in detail below),or to generate antibodies specific for PGC-1α proteins. In a preferredembodiment, a PGC-1α fusion protein expressed in a retroviral expressionvector of the present invention can be utilized to infect bone marrowcells which are subsequently transplanted into irradiated recipients.The pathology of the subject recipient is then examined after sufficienttime has passed (e.g., six weeks).

In another embodiment, a nucleic acid of the invention is expressed inmammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include neuron-specific promoters (e.g., the neurofilamentpromoter; Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci. USA86:5473-5477), albumin promoter (liver-specific; Pinkert et al., 1987,Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton,1988, Adv. Immunol. 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen andBaltimore, 1983, Cell 33:741-748), pancreas-specific promoters (Edlundet al., 1985, Science 230:912-916), and mammary gland-specific promoters(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166). Developmentally-regulatedpromoters are also encompassed, for example the murine hox promoters(Kessel and Gruss, 1990, Science 249:374-379) and the α-fetoproteinpromoter (Camper and Tilghman, 1989, Genes Dev. 3:537-546).

The methods of the invention may further use a recombinant expressionvector comprising a DNA molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner which allowsfor expression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to PGC-1α mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific, or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid, or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes, see Weintraub, H.et al., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to the use of host cells intowhich a PGC-1α nucleic acid molecule of the invention is introduced,e.g., a PGC-1α nucleic acid molecule within a recombinant expressionvector or a PGC-1α nucleic acid molecule containing sequences whichallow it to homologously recombine into a specific site of the hostcell's genome. The terms “host cell” and “recombinant host cell” areused interchangeably herein. It is understood that such terms refer notonly to the particular subject cell but to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aPGC-1α protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory. Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

A host cell used in the methods of the invention, such as a prokaryoticor eukaryotic host cell in culture, can be used to produce (i.e.,express) a PGC-1α protein. Accordingly, the invention further providesmethods for producing a PGC-1α protein using the host cells of theinvention. In one embodiment, the method comprises culturing the hostcell of the invention (into which a recombinant expression vectorencoding a PGC-1α protein has been introduced) in a suitable medium suchthat a PGC-1α protein is produced. In another embodiment, the methodfurther comprises isolating a PGC-1α protein from the medium or the hostcell.

The host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichsequences encoding a polypeptide corresponding to a marker of theinvention have been introduced. Such host cells can then be used tocreate non-human transgenic animals in which exogenous sequencesencoding a marker protein of the invention have been introduced intotheir genome or homologous recombinant animals in which endogenousgene(s) encoding a polypeptide corresponding to a marker of theinvention sequences have been altered. Such animals are useful forstudying the function and/or activity of PGC-1α, for identifying and/orevaluating modulators of PGC-1α polypeptide activity, as well as inpre-clinical testing of therapeutics or diagnostic molecules, for markerdiscovery or evaluation, e.g., therapeutic and diagnostic markerdiscovery or evaluation, or as surrogates of drug efficacy andspecificity.

A transgenic animal of the invention can be created by introducing anucleic acid encoding a polypeptide corresponding to PGC-1α into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the polypeptide of the invention toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986. Similar methods are used for production ofother transgenic animals. A transgenic founder animal can be identifiedbased upon the presence of the transgene in its genome and/or expressionof mRNA encoding the transgene in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying thetransgene can further be bred to other transgenic animals carrying othertransgenes.

To create an homologous recombinant animal, a vector is prepared whichcontains at least a portion of a gene encoding a polypeptidecorresponding to a marker of the invention into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the gene. In a preferred embodiment, the vector isdesigned such that, upon homologous recombination, the endogenous geneis functionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector). Alternatively, the vector canbe designed such that, upon homologous recombination, the endogenousgene is mutated or otherwise altered but still encodes functionalprotein (e.g., the upstream regulatory region can be altered to therebyalter the expression of the endogenous protein). In the homologousrecombination vector, the altered portion of the gene is flanked at its5′ and 3′ ends by additional nucleic acid of the gene to allow forhomologous recombination to occur between the exogenous gene carried bythe vector and an endogenous gene in an embryonic stem cell. Theadditional flanking nucleic acid sequences are of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the vector (see, e.g., Thomas and Capecchi, 1987, Cell51:503 for a description of homologous recombination vectors). Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced gene has homologouslyrecombined with the endogenous gene are selected (see, e.g., Li et al.,1992, Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see, e.g., Bradley, Teratocarcinomas and Embryonic Stem Cells: APractical Approach, Robertson, Ed., IRL, Oxford, 1987, pp. 113-152). Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term. Progeny harboringthe homologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination vectors and homologous recombinantanimals are described further in Bradley (1991) Current Opinion inBio/Technology 2:823-829 and in PCT Publication NOS. WO 90/11354, WO91/01140, WO 92/0968, and WO 93/04169.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.,1991, Science 251:1351-1355). If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut et al. (1997)Nature 385:810-813 and PCT Publication NOS. WO 97/07668 and WO 97/07669.

V. Isolated Nucleic Acid Molecules Used in the Methods of the Invention

The nucleotide sequence of the isolated human PGC-1α cDNA and thepredicted amino acid sequence of the human PGC-1α polypeptide are shownin SEQ ID NOs:1 and 2, respectively. The nucleotide and amino acidsequences of human PGC-1α are also described in GenBank Accession No.GI:29570796. The nucleotide sequence of the isolated human PGC-1α cDNAand the predicted amino acid sequence of the human PGC-1α polypeptideare shown in SEQ ID NOs:45 and 46, respectively. The nucleotide andamino acid sequences of mouse PGC-1α are also described in GenBankAccession No. GI:6679432.

The methods of the invention include the use of isolated nucleic acidmolecules that encode PGC-1α proteins or biologically active portionsthereof, as well as nucleic acid fragments sufficient for use ashybridization probes to identify PGC-1α-encoding nucleic acid molecules(e.g., PGC-1α mRNA) and fragments for use as PCR primers for theamplification or mutation of PGC-1α nucleic acid molecules. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

A nucleic acid molecule used in the methods of the present invention,e.g., a nucleic acid molecule having the nucleotide sequence of SEQ IDNO:1, or a portion thereof, can be isolated using standard molecularbiology techniques and the sequence information provided herein. Usingall or portion of the nucleic acid sequence of SEQ ID NO:1 as ahybridization probe, PGC-1α nucleic acid molecules can be isolated usingstandard hybridization and cloning techniques (e.g., as described inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1 can be isolated by the polymerase chain reaction (PCR) usingsynthetic oligonucleotide primers designed based upon the sequence ofSEQ ID NO:1.

A nucleic acid used in the methods of the invention can be amplifiedusing cDNA, mRNA or, alternatively, genomic DNA as a template andappropriate oligonucleotide primers according to standard PCRamplification techniques. Furthermore, oligonucleotides corresponding toPGC-1α nucleotide sequences can be prepared by standard synthetictechniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, the isolated nucleic acid molecules used inthe methods of the invention comprise the nucleotide sequence shown inSEQ ID NO:1, a complement of the nucleotide sequence shown in SEQ IDNO:1, or a portion of any of these nucleotide sequences. A nucleic acidmolecule which is complementary to the nucleotide sequence shown in SEQID NO:1, is one which is sufficiently complementary to the nucleotidesequence shown in SEQ ID NO:1 such that it can hybridize to thenucleotide sequence shown in SEQ ID NO:1 thereby forming a stableduplex.

In still another preferred embodiment, an isolated nucleic acid moleculeused in the methods of the present invention comprises a nucleotidesequence which is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or more identical to the entire length of thenucleotide sequence shown in SEQ ID NO:1 or a portion of any of thisnucleotide sequence.

Moreover, the nucleic acid molecules used in the methods of theinvention can comprise only a portion of the nucleic acid sequence ofSEQ ID NO:1, for example, a fragment which can be used as a probe orprimer or a fragment encoding a portion of a PGC-1α protein, e.g., abiologically active portion of a PGC-1α protein. The probe/primertypically comprises substantially purified oligonucleotide. Theoligonucleotide typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12 or 15,preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55,60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:1of an anti-sense sequence of SEQ ID NO:1 or of a naturally occurringallelic variant or mutant of SEQ ID NO:1. In one embodiment, a nucleicacid molecule used in the methods of the present invention comprises anucleotide sequence which is greater than 100, 100-200, 200-300,300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000,1000-1100, 1100-1200, 1200-1300, or more nucleotides in length andhybridizes under stringent hybridization conditions to a nucleic acidmolecule of SEQ ID NO:1.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additionalstringent conditions can be found in Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), chapters 7, 9 and 11. A preferred, non-limiting example ofstringent hybridization conditions includes hybridization in 4× sodiumchloride/sodium citrate (SSC), at about 65-70° C. (or hybridization in4×SSC plus 50% formamide at about 42-50° C.) followed by one or morewashes in 1×SSC, at about 65-70° C. A preferred, non-limiting example ofhighly stringent hybridization conditions includes hybridization in1×SSC, at about 65-70° C. (or hybridization in 1×SSC plus 50% formamideat about 42-50° C.) followed by one or more washes in 0.3×SSC, at about65-70° C. A preferred, non-limiting example of reduced stringencyhybridization conditions includes hybridization in 4×SSC, at about50-60° C. (or alternatively hybridization in 6×SSC plus 50% formamide atabout 40-45° C.) followed by one or more washes in 2×SSC, at about50-60° C. Ranges intermediate to the above-recited values, e.g., at65-70° C. or at 42-50° C. are also intended to be encompassed by thepresent invention. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH₂PO₄, and 1.25mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers; washes areperformed for 15 minutes each after hybridization is complete. Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (T_(m)) of the hybrid, where T_(m) is determined accordingto the following equations. For hybrids less than 18 base pairs inlength, T_(m) (° C.)=2(# of A+T bases)+4(# of G+C bases). For hybridsbetween 18 and 49 base pairs in length, T_(m)(°C.)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional preferred,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C., see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (oralternatively 0.2×SSC, 1% SDS).

In preferred embodiments, the probe further comprises a label groupattached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a PGC-1α protein, such as by measuring a levelof a PGC-1α-encoding nucleic acid in a sample of cells from a subjecte.g., detecting PGC-1α mRNA levels or determining whether a genomicPGC-1α gene has been mutated or deleted.

The methods of the invention further encompass the use of nucleic acidmolecules that differ from the nucleotide sequence shown in SEQ ID NO:1due to degeneracy of the genetic code and thus encode the same PGC-1αproteins as those encoded by the nucleotide sequence shown in SEQ IDNO:1. In another embodiment, an isolated nucleic acid molecule includedin the methods of the invention has a nucleotide sequence encoding aprotein having an amino acid sequence shown in SEQ ID NO:2.

The methods of the invention further include the use of allelic variantsof human PGC-1α, e.g., functional and non-functional allelic variants.Functional allelic variants are naturally occurring amino acid sequencevariants of the human PGC-1α protein that maintain a PGC-1α activity.Functional allelic variants will typically contain only conservativesubstitution of one or more amino acids of SEQ ID NO:2, or substitution,deletion or insertion of non-critical residues in non-critical regionsof the protein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human PGC-1α protein that do not have a PGC-1αactivity. Non-functional allelic variants will typically contain anon-conservative substitution, deletion, or insertion or prematuretruncation of the amino acid sequence of SEQ ID NO:2, or a substitution,insertion or deletion in critical residues or critical regions of theprotein.

The methods of the present invention may further use non-humanorthologues of the human PGC-1α protein. Orthologues of the human PGC-1αprotein are proteins that are isolated from non-human organisms andpossess the same PGC-1α activity.

The methods of the present invention further include the use of nucleicacid molecules comprising the nucleotide sequence of SEQ ID NO:1 or aportion thereof, in which a mutation has been introduced. The mutationmay lead to amino acid substitutions at “non-essential” amino acidresidues or at “essential” amino acid residues. A “non-essential” aminoacid residue is a residue that can be altered from the wild-typesequence of PGC-1α (e.g., the sequence of SEQ ID NO:2) without alteringthe biological activity, whereas an “essential” amino acid residue isrequired for biological activity. For example, amino acid residues thatare conserved among the PGC-1α proteins of the present invention andother members of the PGC-1 family are not likely to be amenable toalteration.

Mutations can be introduced into SEQ ID NO:1 by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore predicted non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., glycine, alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in a PGC-1α protein is preferably replaced withanother amino acid residue from the same side chain family.Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a PGC-1α coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forPGC-1α biological activity to identify mutants that retain activity.Following mutagenesis of SEQ ID NO:1 the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined using the assay described herein.

Another aspect of the invention pertains to the use of isolated nucleicacid molecules which are antisense to the nucleotide sequence of SEQ IDNO:1. An “antisense” nucleic acid comprises a nucleotide sequence whichis complementary to a “sense” nucleic acid encoding a protein, e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence. Accordingly, an antisense nucleicacid can hydrogen bond to a sense nucleic acid. The antisense nucleicacid can be complementary to an entire PGC-1α coding strand, or to onlya portion thereof. In one embodiment, an antisense nucleic acid moleculeis antisense to a “coding region” of the coding strand of a nucleotidesequence encoding a PGC-1α. The term “coding region” refers to theregion of the nucleotide sequence comprising codons which are translatedinto amino acid residues. In another embodiment, the antisense nucleicacid molecule is antisense to a “noncoding region” of the coding strandof a nucleotide sequence encoding PGC-1α. The term “noncoding region”refers to 5′ and 3′ sequences which flank the coding region that are nottranslated into amino acids (also referred to as 5′ and 3′ untranslatedregions).

Given the coding strand sequences encoding PGC-1α disclosed herein,antisense nucleic acids of the invention can be designed according tothe rules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to the entire coding region of PGC-1αmRNA, but more preferably is an oligonucleotide which is antisense toonly a portion of the coding or noncoding region of PGC-1α mRNA. Forexample, the antisense oligonucleotide can be complementary to theregion surrounding the translation start site of PGC-1α mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest). Antisense nucleicacid molecules used in the methods of the invention are furtherdescribed above, in section IV.

In yet another embodiment, the PGC-1α nucleic acid molecules used in themethods of the present invention can be modified at the base moiety,sugar moiety or phosphate backbone to improve, e.g., the stability,hybridization, or solubility of the molecule. For example, thedeoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acids (see Hyrup B. et al. (1996)Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms“peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g.,DNA mimics, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of PNAs has been shown to allow forspecific hybridization to DNA and RNA under conditions of low ionicstrength. The synthesis of PNA oligomers can be performed using standardsolid phase peptide synthesis protocols as described in Hyrup B. et al.(1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.93:14670-675.

PNAs of PGC-1α nucleic acid molecules can be used in the therapeutic anddiagnostic applications described herein. For example, PNAs can be usedas antisense or antigene agents for sequence-specific modulation of geneexpression by, for example, inducing transcription or translation arrestor inhibiting replication. PNAs of PGC-1α nucleic acid molecules canalso be used in the analysis of single base pair mutations in a gene,(e.g., by PNA-directed PCR clamping); as ‘artificial restrictionenzymes’ when used in combination with other enzymes, (e.g., S1nucleases (Hyrup B. et al. (1996) supra)); or as probes or primers forDNA sequencing or hybridization (Hyrup B. et al. (1996) supra;Perry-O'Keefe et al. (1996) supra).

In another embodiment, PNAs of PGC-1α can be modified, (e.g., to enhancetheir stability or cellular uptake), by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of PGC-1α nucleic acid molecules can begenerated which may combine the advantageous properties of PNA and DNA.Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNApolymerases), to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup B. et al. (1996) supra). The synthesis of PNA-DNA chimeras can beperformed as described in Hyrup B. et al. (1996) supra and Finn P. J. etal. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chaincan be synthesized on a solid support using standard phosphoramiditecoupling chemistry and modified nucleoside analogs, e.g.,5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can beused as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989)Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide used in the methods of theinvention may include other appended groups such as peptides (e.g., fortargeting host cell receptors in vivo), or agents facilitating transportacross the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl.Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.Sci. USA 84:648-652; PCT Publication No. WO88/09810) or the blood-brainbarrier (see, e.g., PCT Publication No. WO89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) orintercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). Tothis end, the oligonucleotide may be conjugated to another molecule,(e.g., a peptide, hybridization triggered cross-linking agent, transportagent, or hybridization-triggered cleavage agent).

VI. Isolated PGC-1α Proteins and Anti-PGC-1α Antibodies Used in theMethods of the Invention

The methods of the invention include the use of isolated PGC-1αproteins, and biologically active portions thereof, as well aspolypeptide fragments suitable for use as immunogens to raiseanti-PGC-1α antibodies. In one embodiment, native PGC-1α proteins can beisolated from cells or tissue sources by an appropriate purificationscheme using standard protein purification techniques. In anotherembodiment, PGC-1α proteins are produced by recombinant DNA techniques.Alternative to recombinant expression, a PGC-1α protein or polypeptidecan be synthesized chemically using standard peptide synthesistechniques.

As used herein, a “biologically active portion” of a PGC-1α proteinincludes a fragment of a PGC-1α protein having a PGC-1α activity.Biologically active portions of a PGC-1α protein include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the PGC-1α protein, e.g., the amino acidsequence shown in SEQ ID NO:2, which include fewer amino acids than thefull length PGC-1α proteins, and exhibit at least one activity of aPGC-1α protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the PGC-1α protein (e.g.,the N-terminal region of the PGC-1α protein that is believed to beinvolved in the regulation of apoptotic activity). A biologically activeportion of a PGC-1α protein can be a polypeptide which is, for example,25, 50, 75, 100, 125, 150, 175, 200, 250, 300 or more amino acids inlength. Biologically active portions of a PGC-1α protein can be used astargets for developing agents which modulate a PGC-1α activity.

In a preferred embodiment, the PGC-1α protein used in the methods of theinvention has an amino acid sequence shown in SEQ ID NO:2. In otherembodiments, the PGC-1α protein is substantially identical to SEQ IDNO:2, and retains the functional activity of the protein of SEQ ID NO:2,yet differs in amino acid sequence due to natural allelic variation ormutagenesis, as described in detail in subsection V above. Accordingly,in another embodiment, the PGC-1α protein used in the methods of theinvention is a protein which comprises an amino acid sequence at leastabout 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or more identical to SEQ ID NO:2.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the PGC-1α amino acidsequence of SEQ ID NO:2 having 500 amino acid residues, at least 75,preferably at least 150, more preferably at least 225, even morepreferably at least 300, and even more preferably at least 400 or moreamino acid residues are aligned). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated intothe GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentidentity between two nucleotide sequences is determined using the GAPprogram in the GCG software package (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci. 4:11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0 or 2.0U), using a PAM120 weight residue table, agap length penalty of 12 and a gap penalty of 4.

The methods of the invention may also use PGC-1α chimeric or fusionproteins. As used herein, a PGC-1α “chimeric protein” or “fusionprotein” comprises a PGC-1α polypeptide operatively linked to anon-PGC-1α polypeptide. An “PGC-1α polypeptide” refers to a polypeptidehaving an amino acid sequence corresponding to a PGC-1α molecule,whereas a “non-PGC-1α polypeptide” refers to a polypeptide having anamino acid sequence corresponding to a protein which is notsubstantially homologous to the PGC-1α protein, e.g., a protein which isdifferent from the PGC-1α protein and which is derived from the same ora different organism. Within a PGC-1α fusion protein the PGC-1αpolypeptide can correspond to all or a portion of a PGC-1α protein. In apreferred embodiment, a PGC-1α fusion protein comprises at least onebiologically active portion of a PGC-1α protein. In another preferredembodiment, a PGC-1α fusion protein comprises at least two biologicallyactive portions of a PGC-1α protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the PGC-1α polypeptideand the non-PGC-1α polypeptide are fused in-frame to each other. Thenon-PGC-1α polypeptide can be fused to the N-terminus or C-terminus ofthe PGC-1α polypeptide.

For example, in one embodiment, the fusion protein is a GST-PGC-1αfusion protein in which the PGC-1α sequences are fused to the C-terminusof the GST sequences. Such fusion proteins can facilitate thepurification of recombinant PGC-1α.

In another embodiment, this fusion protein is a PGC-1α proteincontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofPGC-1α can be increased through use of a heterologous signal sequence.

The PGC-1α fusion proteins used in the methods of the invention can beincorporated into pharmaceutical compositions and administered to asubject in vivo. The PGC-1α fusion proteins can be used to affect thebioavailability of a PGC-1α substrate. Use of PGC-1α fusion proteins maybe useful therapeutically for the treatment of disorders caused by, forexample, (i) aberrant modification or mutation of a gene encoding aPGC-1α protein; (ii) mis-regulation of the PGC-1α gene; and (iii)aberrant post-translational modification of a PGC-1α protein.

Moreover, the PGC-1α-fusion proteins used in the methods of theinvention can be used as immunogens to produce anti-PGC-1α antibodies ina subject, to purify PGC-1α ligands and in screening assays to identifymolecules which inhibit the interaction of PGC-1α with a PGC-1αsubstrate.

Preferably, a PGC-1α chimeric or fusion protein used in the methods ofthe invention is produced by standard recombinant DNA techniques. Forexample, DNA fragments coding for the different polypeptide sequencesare ligated together in-frame in accordance with conventionaltechniques, for example by employing blunt-ended or stagger-endedtermini for ligation, restriction enzyme digestion to provide forappropriate termini, filling-in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andenzymatic ligation. In another embodiment, the fusion gene can besynthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide). A PGC-1α-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to thePGC-1α protein.

The present invention also pertains to the use of variants of the PGC-1αproteins which function as either PGC-1α agonists (mimetics) or asPGC-1α antagonists. Variants of the PGC-1α proteins can be generated bymutagenesis, e.g., discrete point mutation or truncation of a PGC-1αprotein. An agonist of the PGC-1α proteins can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of a PGC-1α protein. An antagonist of a PGC-1α proteincan inhibit one or more of the activities of the naturally occurringform of the PGC-1α protein by, for example, competitively modulating aPGC-1α-mediated activity of a PGC-1α protein. Thus, specific biologicaleffects can be elicited by treatment with a variant of limited function.In one embodiment, treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein has fewer side effects in a subject relative to treatment withthe naturally occurring form of the PGC-1α protein.

In one embodiment, variants of a PGC-1α protein which function as eitherPGC-1α agonists (mimetics) or as PGC-1α antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of a PGC-1α protein for PGC-1α protein agonist or antagonist activity.In one embodiment, a variegated library of PGC-1α variants is generatedby combinatorial mutagenesis at the nucleic acid level and is encoded bya variegated gene library. A variegated library of PGC-1α variants canbe produced by, for example, enzymatically ligating a mixture ofsynthetic oligonucleotides into gene sequences such that a degenerateset of potential PGC-1α sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of PGC-1α sequencestherein. There are a variety of methods which can be used to producelibraries of potential PGC-1α variants from a degenerate oligonucleotidesequence. Chemical synthesis of a degenerate gene sequence can beperformed in an automatic DNA synthesizer, and the synthetic gene thenligated into an appropriate expression vector. Use of a degenerate setof genes allows for the provision, in one mixture, of all of thesequences encoding the desired set of potential PGC-1α sequences.Methods for synthesizing degenerate oligonucleotides are known in theart (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al.(1984) Annu. Rev. Biochem. 53:323; Itakura et al., (1984) Science198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

In addition, libraries of fragments of a PGC-1α protein coding sequencecan be used to generate a variegated population of PGC-1α fragments forscreening and subsequent selection of variants of a PGC-1α protein. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double stranded PCR fragment of a PGC-1α coding sequencewith a nuclease under conditions wherein nicking occurs only about onceper molecule, denaturing the double stranded DNA, renaturing the DNA toform double stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the PGC-1α protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of PGC-1α proteins. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify PGC-1α variants (Arkin and Yourvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

The methods of the present invention further include the use ofanti-PGC-1α antibodies. An isolated PGC-1α protein, or a portion orfragment thereof, can be used as an immunogen to generate antibodiesthat bind PGC-1α using standard techniques for polyclonal and monoclonalantibody preparation. A full-length PGC-1α protein can be used or,alternatively, antigenic peptide fragments of PGC-1α can be used asimmunogens. The antigenic peptide of PGC-1α comprises at least 8 aminoacid residues of the amino acid sequence shown in SEQ ID NO:2 andencompasses an epitope of PGC-1α such that an antibody raised againstthe peptide forms a specific immune complex with the PGC-1α protein.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofPGC-1α that are located on the surface of the protein, e.g., hydrophilicregions, as well as regions with high antigenicity.

A PGC-1α immunogen is typically used to prepare antibodies by immunizinga suitable subject, (e.g., rabbit, goat, mouse, or other mammal) withthe immunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed PGC-1α protein or a chemicallysynthesized PGC-1α polypeptide. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic PGC-1α preparation induces a polyclonal anti-PGC-1α antibodyresponse.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site which specifically binds(immunoreacts with) an antigen, such as a PGC-1α. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)₂ fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind PGC-1α molecules. Theterm “monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of PGC-1α. A monoclonal antibody composition thustypically displays a single binding affinity for a particular PGC-1αprotein with which it immunoreacts.

Polyclonal anti-PGC-1α antibodies can be prepared as described above byimmunizing a suitable subject with a PGC-1α immunogen. The anti-PGC-1αantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized PGC-1α. If desired, the antibody moleculesdirected against PGC-1α can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-PGC-1α antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int.J. Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique(Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generallyKenneth, R. H. in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); Lerner, E. A.(1981) Yale J. Biol. Med. 54:387-402; Gefter, M. L. et al. (1977)Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a PGC-1α immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds PGC-1α.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-PGC-1α monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature 266:55052; Gefter et al. (1977) supra; Lerner (1981) supra; andKenneth (1980) supra). Moreover, the ordinarily skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line. Preferred immortal cell lines aremouse myeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-×63-Ag8.653 orSp2/O—Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindPGC-1α, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-PGC-1α antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with PGC-1α to thereby isolateimmunoglobulin library members that bind PGC-1α. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J.Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gramet al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al.(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. AcidRes. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. (1990) Nature 348:552-554.

Additionally, recombinant anti-PGC-1α antibodies, such as chimerzic andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the methods of the invention. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described inRobinson et al. International Application No. PCT/US86/02269; Akira, etal. European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison et al. European Patent Application173,494; Neuberger et al. PCT International Publication No. WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559; Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-PGC-1α antibody can be used to detect PGC-1α protein (e.g., in acellular lysate or cell supernatant) in order to evaluate the abundanceand pattern of expression of the PGC-1α protein. Anti-PGC-1α antibodiescan be used diagnostically to monitor protein levels in tissue as partof a clinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

This invention is further illustrated by the following Exemplificationwhich should not be construed as limiting. The contents of allreferences, sequences, Figures, GenBank Accession Numbers, and publishedpatent applications cited throughout this application are herebyincorporated by reference.

EXAMPLES Materials and Methods

The following materials and methods were used for the experimentsdescribed below.

Generation of PGC-1α^(−/−) mice

A targeting plasmid was constructed using genomic DNA fragments derivedfrom Sv129 mouse strain. A loxP site and a neomycin/thymidine kinasecassette flanked by two loxP sites were introduced into the PGC-1αlocus. Embryonic stem (ES) cell (derived from Sv129 strain)electroporation, selection and screening were performed using standardgene targeting techniques. Genomic DNA was isolated fromneomycin-resistant ES cell clones, digested with BamHI and subjected tohybridization using probe L to detect homologous recombination and thepresence of the flox allele (FIG. 9A). Chimeric founders were bred withwild-type C57/Bl6 mice to obtain offspring containing a germ-line PGC-1αflox allele. These mice were subsequently bred with ZP3-cre transgenic(in C57/Bl6 background) mice to generate PGC-1α^(+/−) offspring. The crerecombinase-mediated generation of PGC-1α^(+/−) allele was confirmed bySouthern hybridization using probe L following restriction digestion byBamHI. Heterozygous mice were mated to obtain PGC-1α^(−/−) mice.Genotyping of mice used in this study was performed by PCR with tail DNAas shown in FIG. 9.

Animal Experiments

Mice were maintained on a standard rodent chow or a high-fat dietcontaining 57% fat-derived calories (D12331, Research Diets™) withtwelve-hour light and dark cycles. Plasma glucose and insulinconcentrations were measured from tail blood using glucometer (Lifescan,Johnson & Johnson™) and an insulin ELISA kit (Crystal Chem. Inc.™),respectively.

For diet-induced obesity, body weight was measured weekly for a periodof 4 months. Body fat content was measured using a dual X-rayabsorptiometry (Piximus, Lunar Corporation™) after three months on thehigh-fat diet.

For cold exposure, 6 to 7-week old male mice were individually housed incages kept at 4° C. with free access to food and water. Core bodytemperature was monitored using a rectal thermometer at various timesafter the start of cold exposure. Brown fat was dissected 5 hours aftercold exposure and subjected to gene expression analysis.

Metabolic monitoring was performed using a Comprehensive Lab AnimalMonitoring System (CLAMS, Columbia Instruments) that simultaneouslymeasures whole body oxygen consumption and physical movements forsixteen mice. Mice were acclimated in the monitoring chambers for twodays before the experiment to minimize the changes in housingenvironments. Data was collected every 48 minutes for each mouse over aperiod of three days. Metabolic rate and physical activity were averagedfor the whole study period with the exception of the first five datapoints that tend to be influenced by animal handling at the beginning ofstudies.

Histological Analysis

Tissues were dissected and fixed in 4% paraformaldehyde overnight andrinsed with phosphate-buffered saline. Brown fat was subsequentlydehydrated and embedded in plastic (JB-4, Electron Microscopy Sciences™)and sectioned at 1.5 μm for Hematoxylin and Eosin (H&E) staining. Livertissue was dehydrated in ethanol, paraffin-embedded, and sectioned at 4μm for H&E staining.

Brain tissue for neuropathological examination was prepared from 6 and12-week old wild-type and PGC-1α null mice. Tissues were fixed in situby intracardiac perfusion with 15 ml PBS followed by 30 ml 4%paraformaldehyde (PFA) in phosphate buffer solution (PBS). Brains wereremoved, postfixed in 4% PFA overnight at 4° C. and embedded inparaffin. Coronal sections (6 μm) were stained both conventionally withH&E, luxol fast blue (H&E+LFB), and 0.1% cresyl echt violet (Niss1), andimmunohistochemically with antibodies against glial fibrillary acidicprotein (GFAP, polyclonal antibody 1:200; Dako™, Hamburg, Germany) as anastrocyte marker and against neurofilament 200 kD (monoclonal antibody1:50; Sigma™) to label axons.

RNA and Protein Analysis

Total RNA was isolated from cultured hepatocytes or tissues using Trizolreagents (Invitrogen™). For hybridization, 10-20 μs of RNA samples wereseparated on a formaldehyde gel, transferred to nylon membrane and thenhybridized with gene-specific probes. For real-time PCR analysis, RNAsamples were reverse-transcribed and used in quantitative PCR reactionsin the presence of a fluorescent dye (Cybergreen™, Bio-rad™). Relativeabundance of mRNA was calculated after normalization to 18S ribosomalRNA. Sequences for the primers used in this study were shown in Table 1.

TABLE 1 List of primers used in real-time PCR analysis. SEQ SEQ GeneForward primer ID NO. Reverse primer ID NO. PGC-1αagccgtgaccactgacaacgag  3 gctgcatggttctgagtgctaag 22 PGC-1βcgctccaggagactgaatccag  4 cttgactactgtctgtgaggc 23 PEPCKcatatgctgatcctgggcataac  5 caaacttcatccaggcaatgtc 24 G6Paseacaccgactactacagcaacag  6 cctcgaaagatagcaagagtag 25 UCP1ggcattcagaggcaaatcagct  7 caatgaacactgccacacctc 26 Cox7a1gtctcccaggctctggtccg  8 ctgtacaggacgttgtccattc 27 Ndufb5tccgaagactgtcgctcctgtg  9 tatgttcaccagtgttatgcca 28 CKmt2ggtacgcactggccgaagcatc 10 tgatcctgctccgtcatctcag 29 H-FABPgtcggtacctggaagctagtggac 11 gatctctgtgttcttgaaggtac 30 Atp5Jgttctgcagaggatcttcaggc 12 gtcctccagatgcctgtcgctt 31 Dio2cagtgtggtgcacgtctccaatc 13 tgaaccaaagttgaccaccag 32 UCP2caggtcactgtgcccttacca 14 cactacgttccaggatcccaa 33 18Sagtccctgccctttgtacaca 15 cgatccgagggcctcacta 34 LDH2cactgtagtgggcgttggacaa 16 cggccacaattttcggagtctg 35 Cox6a1caacgtgttcctcaagtcgcgg 17 gccaggttctctttactcatc 36 NF-Hgctggacagtgagctgagaaac 18 caaagccaatccgacactcttc 37 NF-Mgatgagctacacgctggactcg 19 tgtaggaggaggacacggtgct 38 MOBPactccaagcgtgagatcgtggac 20 ggacgcagctggctggtgcttg 39 ATP1a1tcatcgtagccaacgtgccag 21 gtcttgtctgagcagatggtag 40

For the detection of PGC-1α protein, nuclear extracts (80 μg) preparedfrom brown fat were analyzed by immunoblotting using mouse polyclonalantibodies raised against purified C-terminus of PGC-1α. Muscle AMPK wasdetected with antibodies that recognize total AMPK (#07-181, UpstateBiotechnology™) or phosphorylated AMPK (2531, Cell SignalingTechnology™). ACC phosphorylation was detected using a phospho-ACCspecific antibody (3661, Cell Signaling Technology™).

Primary Hepatocytes

Primary hepatocytes were isolated following perfusion of whole liverfirst with perfusion buffer (Hank's Balanced Saline, HBSS) and thencollagenase solution (HBSS with 1% BSA and 0.05% collagenase) for 10minutes. Dispersed cells were resuspended and seeded ontocollagen-coated plates in DMEM supplemented with 10% fetal bovine serumin the presence of 1 mM sodium pyruvate, 1 μM dexamethasone (dex) and 50nM insulin. Two hours after plating, the medium was changed to amaintenance medium containing DMEM supplemented with 0.1% BSA and 1 mMsodium pyruvate. For hormonal treatments, hepatocytes were cultured inminimal media (DMEM with 0.1% BSA) for 40 hours and then treated with0.2 μM or 1 μM of a combination of forskolin and dex for 3 or 6 hoursbefore RNA isolation.

For adenoviral infection, hepatocytes were incubated with varying titersof adenovirus expressing either GFP or C/EBPβ for 3 hours and thenmaintained in starvation media for 24 hours. Infected cells were treatedwith 0.2 μM forskolin and 0.1 μM dex for 3 hours before RNA isolation.

For respiration measurements, hepatocytes were cultured in the presenceof 0.2 μM dex overnight and then removed from plates by incubating withthe trypsin/EDTA solution. Oxygen consumption was measured essentiallyas previously described (Fan, M., et al. (2004) Genes Dev 18, 278-289;Wu, Z., (1999) Cell 98, 115-124). Measurements were performed in thepresence of 25 mM glucose, 1 mM pyruvate and 2% BSA.

Insulin/Glucose/Pyruvate Tolerance Tests

For insulin tolerance test, high-fat fed mice were fasted for 4 hoursbefore receiving an intraperitoneal injection of insulin at 0.8 mU/kg.Plasma glucose levels were measured from tail blood before or 15, 30,45, 60, 100 minutes after insulin infusion.

For glucose tolerance test, high-fat fed mice were fasted overnight (14hours) and injected intraperitoneally with a glucose solution (preparedin saline) at 2 g/kg. Plasma glucose levels were measured from tailblood before or 15, 30, 45, 60, 90, 180 minutes after glucose infusion.

Pyruvate tolerance test was performed as described (Miyake, K., et al.(2002) J Clin Invest 110, 1483-1491). Briefly, three-month old male micewere fasted for 14 hours before receiving an intraperitoneal dose ofpyruvate (in saline) at 2 g/kg. Plasma glucose levels were determined asindicated above.

Transient Transfection

Mouse H2.35 hepatoma cells (CRL-1995, ATCC) were maintained in DMEMsupplemented with 4% fetal bovine serum in the presence of 0.2 μM dex.For transfection, 100 ng of reporter plasmid (Gal-C/EBPβ) wastransiently transfected using Superfect™ (Qiagen™) in the presence of500 ng of vector or pcDNA3-PGC-1α. Luciferase activity was measured 40hours following transfection.

Example 1 Generation of PGC-1α Null Mice

To generate mouse strains deficient in PGC-1α by homologousrecombination, a targeting plasmid was constructed flanking exons 3 to 5of the PGC-1α gene with loxP sites (FIG. 9A). These three exons encode ahighly conserved region in PGC-1α, including the LXXLL motif thatmediates its interaction with many nuclear receptors. PGC-1α^(+/−) micewere generated through transgenic expression of cre recombinase underthe control of ZP3 promoter, which is transiently activated duringoocyte development (Lewandoski, M. et al. (1997) Curr Biol 7, 148-151).PGC-1α^(−/−) mice were obtained from offspring of heterozygous breedingpairs. Homologous recombination and cre-mediated excision events wereconfirmed by hybridization and PCR analysis of genomic DNA isolated fromPGC-1α^(+/+), PGC-1α^(+/−) and PGC-1α^(−/−) mice (FIGS. 9B-D). Analysisof PGC-1α expression revealed that its mRNA was absent in skeletalmuscle and liver from PGC-1α^(−/−) mice and reduced to approximately 50%in PGC-1α^(+/−) mice as revealed by RNA hybridization and real-time PCRanalysis (FIG. 1A). As expected, no PGC-1α protein was detected in thenuclear extract prepared from PGC-1α^(−/−) brown fat (FIG. 1B).

Pups lacking PGC-1α were born at the expected Mendelian ratio,suggesting that PGC-1α is dispensable for embryonic development.However, only half of PGC-1α^(−/−) pups survive early postnatal periodand grow into adults (Table 2).

TABLE 2 Genotypes of offspring from heterozygous breeding pairs atvarious stages. Genotype Total PGC-1α^(+/+) PGC-1α^(+/−) PGC-1α^(−/−)Day 1 pups  17 (23%)  38 (52%) 18 (25%) 73 Weaning 135 (29%) 264 (57%)65 (14%) 464 (day 21) PGC-1α^(−/−) mice weigh approximately 10-15% lessthan the PGC-1α^(+/+) and PGC-1α^(+/−) littermates at two months of ageand are fertile. Hematoxylin/eosin (H&E) staining of tissue sectionsrevealed normal histology in several tissues including heart, skeletalmuscle, pancreas and liver (FIGS. 1C-D). In contrast, PGC-1α^(−/−) brownfat appeared abnormal, with abundant accumulation of large lipiddroplets (FIG. 1E-F), a feature commonly associated with impairedthermogenic function. Electron microscopy studies revealed no obviouschanges in the abundance and morphology of mitochondria in brown fat andliver.Measurement of plasma and liver lipid levels shows no significantdifference between wild-type and PGC-1α null mice in the fed state whilethe triglyceride content is lower in the liver of null mice (Table 3).

TABLE 3 Plasma and liver lipids and liver glycogen content in wild type.Shown is Mean ± SEM. Lipids/ glycogen PGC-1α^(+/+) PGC-1α^(−/−) Chow-fedPlasma Fed 78.6 ± 9.0 75.7 ± 5.4 triglyceride 24-hr 75.7 ± 9.3 68.3 ±9.7 (mg/dL) fasting 48-hr 56.4 ± 5.7 54.6 ± 5.9 fasting Plasma Fed 68.0± 3.8 65.3 ± 2.5 cholesterol 24-hr 81.3 ± 3.6 72.1 ± 1.9 (mg/dL) fastingPlasma free Fed  0.97 ± 0.09  1.09 ± 0.04 fatty acids 24-hr  1.77 ± 0.16 1.56 ± 0.06 (mM) fasting Liver Fed  3.4 ± 0.7  3.8 ± 0.9 triglyceride24-hr 56.5 ± 4.1       34.9 ± 6.7 (p = 0.024) (mg/g) fasting Liver Fed 7.6 ± 0.6      10.3 ± 1.3 (p = 0.09) glycogen 24-hr  2.0 ± 0.2      2.8 ± 0.4 (p = 0.11) (mg/g) fasting High-fat fed Plasma Fed 94.0 ±9.0 77.0 ± 8.9 triglyceride (mg/dL) Plasma Fed 129.9 ± 8.3       106.2 ±4.6 (p = 0.016) cholesterol (mg/dL)

Example 2 PGC-1α Plays a Critical Role In Hormone-InducedGluconeogenesis

PGC-1α has been shown to influence glucose metabolism in muscle andliver. The expression of PGC-1α itself is induced in liver in responseto fasting, a metabolic state characterized by active glycogenolysis,gluconeogenesis and fatty acid β-oxidation (Aoki, T. T. (1981) Prog ClinBiol Res 67, 161-177). To examine the requirement for PGC-1α in theregulation of glucose metabolism, measured blood glucose and insulinlevels were measured in mice under various nutritional states. There isno difference in plasma glucose levels in PGC-1α^(+/+), PGC-1α^(+/−) andPGC-1α^(−/−) mice when food is provided ad libitum (FIG. 2A). Micedeficient in PGC-1α, however, develop mild hypoglycemia after 24 hoursof fasting. Examination of blood insulin levels revealed that althoughPGC-1α^(−/−) mice are able to maintain euglycemia in the fed state, theydo so with reduced circulating insulin concentrations (FIG. 2B). Incontrast, no reduction in insulin levels was observed in PGC-1α^(−/−)mice in the fasted state. Blood glucose levels tend to be lower in thePGC-1α^(−/−) mice after prolonged fasting (48 hours) although thedifference does not reach statistical significance.

PGC-1α has been shown to control the expression of genes involved inmitochondrial fatty acid β-oxidation and oxidative phosphorylation invarious cell types including hepatocytes. Proper mitochondrialrespiration is necessary to generate the ATP that supports the enzymaticfunction of the gluconeogenic pathway. To determine the effects ofPGC-1α deficiency on mitochondrial respiration, an oxygen electrode wasused to measure oxygen consumption in isolated hepatocytes. Total oxygenconsumption rate is reduced 17% in PGC-1α^(−/−) hepatocytes compared towild-type controls (FIG. 2C). Respiration due to mitochondrial protonleak is also reduced approximately 20% in the PGC-1α deficienthepatocytes (FIG. 2C). These data illustrate that mitochondrial functionis impaired in the hepatocytes from PGC-1α^(−/−) mice.

Hepatic gluconeogenesis is of major importance in the fasted state(Hanson, R. W., and Reshef, L. (1997) Annu Rev Biochem 66, 581-611;Pilkis, S. J., and Granner, D. K. (1992) Annu Rev Physiol 54, 885-909)and is controlled by PGC-1α in gain of function experiments (Herzig, S.,et al. (2001) Nature 413, 179-183; Yoon, J. C., et al. (2001) Nature413, 131-138). The induction of PGC-1α and gluconeogenic genes, such asphosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase(G6Pase), are mediated by a rise in the circulating concentrations ofcounter-regulatory hormones, such as glucagon and glucocorticoids, and afall in insulin levels. To determine whether PGC-1α is essential forexpression of this program, primary hepatocytes were isolated fromPGC-1α^(+/+) and PGC-1α^(−/−) mice and examined the expression of thePEPCK and G6Pase genes in response to hormonal treatments. Basal levelsof PEPCK and G6Pase mRNA were similar in PGC-1α^(+/+) and PGC-1α^(−/−)hepatocytes, as revealed by RNA hybridization and quantitative real-timePCR analysis (FIG. 2D). Following treatments with glucocorticoid andforskolin, PGC-1α^(+/+) hepatocytes exhibit a robust and dose-dependentincrease in PEPCK and G6Pase mRNA (FIG. 2D). In contrast, the inductionof PEPCK and G6Pase mRNA expression is greatly diminished in hepatocytesisolated from PGC-1α^(−/−) mice at all treatment conditions examined.These results clearly demonstrate that PGC-1α is an essential mediatorof transcriptional activation of gluconeogenic genes in response tohormonal stimulation in isolated hepatocytes.

To determine whether PGC-1α is necessary for gluconeogenesis in vivo,blood glucose levels in mice were examined following intra-peritonealinjection of pyruvate, an important substrate for this pathway.Consistent with defects in gluconeogenic gene expression andmitochondrial function in the PGC-1α^(−/−) hepatocytes, mice lackingPGC-1α have greatly reduced ability to convert pyruvate into glucosecompared to wild-type littermates (FIG. 2E).

Example 3 Constitutive Activation of the Hepatic Gluconeogenic ProgramIn Vivo in the Absence of PGC-1α

The nutritional regulation of PEPCK and G6Pase genes under fed andfasted states in the mice was examined. As expected, wild-type miceactivate multiple adaptive metabolic changes in response to fasting(FIG. 3A), as shown by increased expression of gluconeogenic genes andthose involved in fatty acid oxidation (MCAD), ketone body synthesis(mitochondrial HMG-CoA synthase) and bile acid synthesis (Cyp7a1) (Rhee,J., et al. (2003) Proc Natl Acad Sci USA 100, 4012-4017; Shin, D. J., etal. (2003) J Biol Chem 278, 50047-50052). Surprisingly, the expressionof PEPCK, G6Pase, HMG-CoA synthase and MCAD is similar in PGC-1α^(+/+)and PGC-1α^(−/−) liver in the fasted state (FIG. 3A). More unexpectedly,the mRNA levels of PEPCK, G6Pase and HMG-CoA synthase are greatlyelevated in PGC-1α^(−/−) mouse liver under fed conditions (FIG. 3A).Notably, expression of G6Pase in the fed state is near the level that isusually seen in fasted liver. These results indicate that althoughPGC-1α is not required for the expression of gluconeogenic and ketogenicgenes, this coactivator is essential for proper nutritional regulationof this response. Since basal levels of PEPCK and G6Pase expression aresimilar in untreated primary hepatocytes in the presence or absence ofPGC-1α (FIG. 2D), this indicates that systemic signals are probablyresponsible for the constitutive activation of these genes under fedconditions in mice lacking PGC-1α.

The aberrant activation of PEPCK and G6Pase expression is likely due todysregulation of one or more transcription factors that can regulate thePEPCK and G6Pase genes in the absence of PGC-1α. To assess thispossibility, mRNA levels of several transcription factors known to haveat least some connection with this process were examined. As shown inFIG. 3B, the expression of HNF4α and PPARα, key regulators of hepaticmetabolism in the fasted state, was comparable in PGC-1α^(+/+) andPGC-1α^(−/−) liver in the fed state, although the induction of HNF4α inresponse to fasting is blunted in the absence of PGC-1α. The mRNA levelsfor FOXO1 and GR remain unchanged between genotypes in both fed andfasted states. As expected, the expression of sterol response elementbinding protein 1c (SREBP1c), a central regulator of hepatic lipogenesis(Horton, J. D., et al. (2002) J Clin Invest 109, 1125-1131), issuppressed in response to fasting in both PGC-1α^(+/+) and PGC-1α^(−/−)mice. This indicates that although the expression of gluconeogenic andketogenic genes is dysregulated in the absence of PGC-1α, thenutritional regulation of SREBP1c is still intact. In contrast, there isa striking increase in the abundance of C/EBPβ mRNA in PGC-1α^(−/−)mouse liver, precisely mirroring the aberrant expression of PEPCK andG6Pase in the fed state (FIG. 3A). C/EBPβ is normally induced in thefasted state in wild-type animals but is abnormally activated in the fedstate in the null mice. C/EBPδ is also induced in the fed liver lackingPGC-1α while C/EBPα is expressed normally with respect to PGC-1αgenotypes. The aberrant induction of C/EBPβ and C/EBPδ, however, isabsent when PGC-1α-deficient hepatocytes are grown in cell culture,indicating that systemic signals are likely responsible for theirincreased expression (FIG. 3C). These results indicate that alteredC/EBP transcription factor activities, particularly C/EBPβ, may play arole in the constitutive activation of gluconeogenic gene expressionseen in the PGC-1α deficient mouse liver.

Example 4 PGC-1α-Independent Activation of Gluconeogenic Genes by C/EBPβ

C/EBPβ is a transcription factor that belongs to the basicleucine-zipper family and has been shown to modulate the activity of thetransfected PEPCK promoter in response to gluconeogenic hormones(Croniger, C. et al. (1998) J Biol Chem 273, 31629-31632; Park, E. A. etal. (1993) J Biol Chem 268, 613-619; Roesler, W. J. (2001) Annu Rev Nutr21, 141-165). Pups lacking C/EBPβ develop severe hypoglycemia and halfof them died shortly after birth due to a failure to activate hepaticgluconeogenic gene expression and glucose production (Croniger, C. etal. (1997) J Biol Chem 272, 26306-26312; Liu, S. et al. (1999) J ClinInvest 103, 207-213). The effects of C/EBPβ on the endogenousgluconeogenic genes have not been studied. Moreover, a functionalinteraction between C/EBPβ and PGC-1α has not been examined to date. Asshown in FIG. 3D, PGC-1α does not coactivate C/EBPβ in transienttransfection assays. Whether C/EBPβ could modulate expression ofendogenous genes of gluconeogenesis and whether it could do so in theabsence of PGC-1α was next examined. Primary hepatocytes were isolatedfrom wild-type and PGC-1α deficient mice, infected with a recombinantadenovirus expressing a control GFP protein or C/EBPβ and examined forthe expression of PEPCK and G6Pase. As shown in FIG. 3E, adenoviralmediated expression of C/EBPβ activates the transcription of the G6Pasegene approximately 17-fold in wild-type hepatocytes. PEPCK mRNA level isalso elevated 1.8-fold in response to ectopic C/EBPβ expression.Importantly, induction of gluconeogenic gene expression by C/EBPβ wasalso observed in the PGC-1α^(−/−) cells with a 12-fold increase inG6Pase mRNA and approximately 3-fold induction of PEPCK mRNA. Theabsolute levels of these mRNAs are somewhat higher in hepatocytes withan intact PGC-1α gene. These results demonstrate that C/EBPβ is able toturn on gluconeogenic gene expression in a PGC-1α-independent manner andindicate that elevated C/EBPβ in PGC-1α^(−/−) animals may be at leastpartially responsible for the inappropriate activation of thosegluconeogenic genes in the fed state.

Example 5 PGC-1α^(−/−) Mice are Resistant to Diet-Induced Obesity

The well-established role of PGC-1α in stimulating mitochondrialrespiration, as well as the reduction of respiration in PGC-1α deficienthepatocytes (FIG. 2C) and abnormal brown fat morphology (FIG. 1D) allsuggest that the null mice may be prone to the development of obesitydue to reduced energy expenditure. To critically assess this, theanimals were fed a high-fat diet containing 58% of calories derived fromfat. As shown in FIG. 4A, and as expected, wild-type mice gainsubstantial body weight throughout the course of high-fat feeding. Incontrast, PGC-1α^(−/−) mice are surprisingly resistant to diet-inducedobesity (FIG. 4A). Analysis of body fat content using a dual energyX-ray absorptiometry (DEXA) scanner revealed that PGC-1α^(−/−) mice areremarkably leaner (22.6±2.4% body fat, n=6) than the PGC-1α^(+/+)controls (39.8±2.8% body fat, n=5) after 16 weeks of high-fat feeding(FIGS. 4B-C). In fact, a small but significant decrease in body fatcontent was observed in PGC-1α^(−/−) mice fed a chow diet (FIG. 4C). Asexpected from their obesity, high-fat fed PGC-1α^(+/+) mice developedinsulin resistance as indicated by elevated fasting glucose and insulinconcentrations and in vivo insulin tolerance test (FIGS. 4D-E).PGC-1α^(−/−) mice, however, display significantly enhanced insulinsensitivity and improved glycemic control in a glucose tolerance test(FIGS. 4E-F) compared to the control mice. Similar results were seen inPGC-1α^(−/−) mice when maintained on a normal rodent chow. These dataclearly indicate that PGC-1α null mice are resistant to obesity causedby high-fat feeding and are protected from developing insulin resistanceand glucose intolerance that ordinarily accompanies this obesity.

The two major arms of energy balance were also examined: energy intakeas measured by food intake and energy expenditure. Both PGC-1α^(+/+) andPGC-1α^(−/−) mice show similar food intake at various time points duringpostnatal development and high-fat feeding (FIG. 5A). In contrast,energy expenditure, measured as O₂ consumption, is approximately 23%higher in PGC-1α^(−/−) mice throughout a three-day period of metabolicmonitoring (FIG. 5B). Thus, the resistance to diet-induced obesity inPGC-1α^(−/−) mice correlates with a substantial increase in energyexpenditure.

Example 6 Reduced Thermogenic Capacity and Hyperactivity in PGC-1α^(−/−)Mice

Two key components of energy expenditure are adaptive thermogenesis andphysical activity. To determine whether increased oxygen consumption inPGC-1α^(−/−) mice is due to enhanced thermogenesis, the thermogeniccapacity of these animals was examined with a standard cold challenge.Resting body temperature is similar between PGC-1α^(+/+) andPGC-1α^(−/−) mice (FIG. 5C, t=0). 6-week old mice were exposed to 4° C.and their core body temperature monitored. Both PGC-1α^(+/+) andPGC-1α^(−/−) mice respond to cold temperature by increasing frequency ofshivering. In contrast to wild-type littermates, which are able to keepbody temperature around 36.5° C. after an initial drop of approximately1.5° C., PGC-1α^(−/−) mice display striking sensitivity to the coldtemperature (FIG. 5C). Body temperature of PGC-1α^(−/−) mice drops to33.5° C. within 3 hours and the hypothermia becomes lethal if theexposure of the null mice is extended beyond 6 hours. Defense of bodytemperature in mice is mainly the function of brown adipose tissue,which generates heat due in large part to abundant expression of themitochondrial uncoupler, UCP1 (Bouillaud, F. et al. (1985) Proc NatlAcad Sci USA 82, 445-448; Jacobsson, A., et al. (1985) J Biol Chem 260,16250-16254). Analysis of brown fat gene expression revealed that theinduction of UCP1 in the null mice was reduced to approximately 45% ofthe wild-type level while the induction of type 2 iodothyroninedeiodinase (D2) mRNA was reduced nearly 50% in PGC-1α^(−/−) micecompared to controls (FIG. 5D). The expression of PGC-113 is unchangedwhile mRNAs encoding several enzymes involved in mitochondrial electrontransport and fatty acid oxidation are reduced in PGC-1α^(−/−) brownfat. These data indicate that the increased energy expenditure in thePGC-1α^(−/−) mice is not due to increased thermogenesis in theseanimals; on the contrary, the mice are hypothermic when challenged.

Skeletal muscle is a major tissue involved in energy expenditure invivo. To determine whether PGC-1α is also required for normalmitochondrial function in skeletal muscle, mitochondrial gene expressionin quadriceps muscle from wild-type and PGC-1α null mice was examined.mRNA levels for a large number of genes involved in fatty acid oxidationand mitochondrial function are reduced 30-60% in PGC-1α^(−/−) mice,including those involved in intracellular fatty acid trafficking, theKrebs Cycle, electron transport (Ndufb5 and Cox7a1), ATP synthesis(Atp5j) and mitochondrial protein translation (FIG. 5E). Interestingly,the expression of ERRα, a direct target of PGC-1α and an importantmediator of PGC-1α action, is also reduced in the absence of PGC-1αwhile PGC-1β mRNA level remains largely unchanged (FIG. 5E).

Impaired mitochondrial energy metabolism might be expected to negativelyaffect ATP/AMP ratios. Consistent with this, AMP-activated proteinkinase (AMPK), a key component of cellular energy-sensing pathways(Carling, D. (2004) Trends Biochem Sci 29, 18-24), is strongly activatedin PGC-1α deficient skeletal muscle. The levels of phosphorylated AMPKand acetyl-CoA carboxylase (ACC), a known substrate for activated AMPK,are significantly increased in PGC-1α^(−/−) muscle (FIG. 5F). These dataclearly indicate that PGC-1α is essential for normal mitochondrial geneexpression and energy metabolism in skeletal muscle.

The lack of an increase in thermogenesis or mitochondrial geneexpression in the mutant animals suggested that the higher metabolicrate in PGC-1α deficient mice might be caused by altered levels ofphysical activity. To assess this, the frequency of animal movements wasmonitored and quantitated using the Comprehensive Lab Animal MonitoringSystem (CLAMS), which is capable of simultaneously recording whole bodyoxygen consumption and physical activity. As shown in FIGS. 6A-B, ahigher oxygen consumption rate in PGC-1α^(−/−) mice is accompanied byprofound hyperactivity as indicated by increased physical movement.PGC-1α deficient mice are hypermetabolic and hyperactive compared towild-type controls both during daytime and at night. In fact, the PGC-1αnull mice displayed a 40% increase in the frequency of random movementsduring the monitoring period (FIG. 6C). These results strongly indicatethat the increase in energy expenditure seen in PGC-1α^(−/−) mice is dueto the hyperactivity displayed by the null animals.

Example 7 Striatal Degeneration in PGC-1α Null Mouse Brain

Hyperactivity in PGC-1α^(−/−) mice could result from altered circulatinghormones and/or signals that originated from the central nervous system.Measurements of several hormones known to influence animal movement,including thyroid hormone and catecholamines, showed no significantalterations (Table 4).

TABLE 4 Concentrations of circulating hormones in wild type and PGC-1αnull mice. Hormones PGC-1α^(+/+) PGC-1 α^(−/−) Triiodothyronine (ng/ml)0.43 ± 0.06 0.44 ± 0.04 (n.s.) Leptin (ng/ml) 0.86 ± 0.22   0.25 ± 0.09(p = 0.01) Glucagon (pg/ml) 61 ± 23 52 ± 14 (n.s.) Corticosterone(ng/ml) 231 ± 35  196 ± 27 (n.s.)  Epinephrine (pg/ml) 485 ± 144 768 ±304 (n.s.) Norepinephrine (pg/ml) 1484 ± 280  1042 ± 291 (n.s.) 

Besides a simple increase in total movement, it was observed that PGC-1αnull mice display behavioral changes that are characteristic of certainneurological disorders, including stimulus-induced myoclonus,exaggerated startle responses, dystonic posturing and frequent limbclasping (FIG. 6D). These findings are consistent with lesions in thestriatum, the brain area that is affected in certain neurologicaldiseases characterized by disorders of movement, including Huntington'sdisease.

Neuropathology in the PGC-1α^(−/−) mouse brain was assessed byhistological analysis. The overall brain anatomy appeared similar in thePGC-1α^(+/+) and PGC-1α^(−/−) mice. However, a striking spongiformpattern of lesions was found predominantly in the striatum ofthree-month old PGC-1α^(−/−) mice (FIG. 7). The number and size oflesions decrease from the dorsal to the ventral side and also from thelateral to the medial parts of the striatum. Occasionally, much smallerand less abundant lesions were also found in the cortex, especially incortical layer V/VI of the motor cortex, nucleus accumbens, thalamus,substantia nigra, hippocampus and the mammalliary body. The spongiformlesions in the striatum and brain stem were associated with gliosis, asindicated by strong immunoreactivity for glial fibrillary associatedprotein (GFAP), a hallmark for reactive astrocytes (FIGS. 7E-F). Noreactive astrocytes were found in the minor lesions in other brainareas. To determine if the lesions were mainly affecting the whitematter, brain sections of PGC-1α^(+/+) and PGC-1α^(−/−) mice werestained with Luxol fast blue for myelin. As seen in FIGS. 7A-D, lesionsare predominantly associated with the white matter and rarely with thegrey matter. The overall neuronal density appeared similar in wild-typemice and PGC-1α null mice, although neurons containing vacuoles inPGC-1α^(−/−) mouse brain were occasionally observed. Immunostainingusing a neurofilament-heavy chain (NF220) antibody showed that striatalneurons have lost NF220-positive neurites in the absence of PGC-1α(FIGS. 7G-H). In fact, the spongiform lesions appear to arise from theloss of axons in the striatal area in PGC-1α^(−/−) mouse brain. Theseresults clearly demonstrate that PGC-1α is required for normal brainfunction and that loss of PGC-1α leads to neuronal degeneration inspecific brain areas, most prominently in the striatum.

PGC-1α has been shown to regulate oxidative metabolism and biologicalprograms associated with increased oxidative capacity in atissue-specific manner. Analysis of gene expression in the brains ofwild-type and mutant mice by real-time PCR revealed that mRNA levels ofmany mitochondrial genes are reduced in mutants (FIG. 8A).Interestingly, the expression of several brain-specific genes notinvolving mitochondrial function, including those encoding neurofilamentproteins (NF—H and NF-M), myelin-associated oligodendrocyte basicprotein (MOBP) and Na⁺/K⁺ ATPase (ATP1a2) is also significantly reducedin the PGC-1α null brain compared to wild-type controls (FIG. 8B). Incontrast, mRNA encoding another sodium pump subunit, ATP1a1, is notaltered. These results indicate that, in addition to its key role in theregulation of mitochondrial gene expression, PGC-1α may also haveimportant function in the control of neuronal gene expression andfunction. In fact, primary striatal neurons isolated from PGC-1α^(−/−)mouse embryos display a severe impairment in neurite growth. Striatalneurons lacking PGC-1α have greatly reduced branches of neurites whereaswild-type neurons exhibit robust neurite outgrowth and form an extensivenetwork in culture.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-43. (canceled)
 44. A method for treating or preventing a neurologicaldisease or disorder in a subject comprising the step of administering tosaid subject a peroxisome proliferator-activated receptor gammacoactivator 1 alpha (PGC-1α) modulator, wherein the PGC-1α modulatorincreases PGC-1α expression or activity, and wherein the modulator is aPGC-1α polypeptide comprising the amino acid sequence of SEQ ID NO: 2 ora portion thereof, such that said polypeptide or portion thereofmaintains the ability to modulate one or more of the followingbiological activities: mitochondrial function; the activity orexpression of a mitochondrial gene selected from the group consisting ofLDH2, Ndufb5, COX6a1, and ATP5j; the activity or expression of aneuronal gene selected from the group consisting of NF—H, NF-M, MOBP,ATPα1, and ATP1α2; lesion formation; neurite formation; neurite growth;neuronal degeneration; body weight; energy expenditure; gluconeogenesis;and interaction with nuclear hormone receptors, such that theneurological disease or disorder is treated or prevented.
 45. The methodof claim 44, wherein the PGC-1α polypeptide or portion thereof comprisesan amino acid sequence which is at least 90 percent identical to theamino acid sequence of SEQ ID NO:
 2. 46. The method of claim 44, whereinthe PGC-1α polypeptide or portion thereof is encoded by a nucleic acidmolecule which hybridizes to a complement of a nucleic acid moleculeconsisting of SEQ ID NO:1 at 6×SSC at 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 65° C.
 47. The method of claim 44,wherein the PGC-1α polypeptide or portion thereof further comprises aheterologous polypeptide.
 48. A method for treating or preventing aneurological disease or disorder in a subject comprising the step ofadministering to said subject a PGC-1α modulator, wherein the PGC-1αmodulator increases PGC-1α expression or activity, and wherein themodulator is a PGC-1α nucleic acid comprising the nucleotide sequence ofSEQ ID NO:1, or a portion thereof, such that said polypeptide or portionthereof maintains the ability to modulate one or more of the followingbiological activities: mitochondrial function; the activity orexpression of a mitochondrial gene selected from the group consisting ofLDH2, Ndufb5, COX6a1, and ATP5j; the activity or expression of aneuronal gene selected from the group consisting of NF—H, NF-M, MOBP,ATPα1, and ATP1α2; lesion formation; neurite formation; neurite growth;neuronal degeneration; body weight; energy expenditure; gluconeogenesis;and interaction with nuclear hormone receptors, such that theneurological disease or disorder is treated or prevented.
 49. The methodof claim 48, wherein the PGC-1α nucleic acid or portion thereofcomprises a nucleic acid sequence which is at least 90 percent identicalto the nucleic acid sequence of SEQ ID NO:1.
 50. The method of claim 48,wherein the PGC-1α nucleic acid or portion thereof further encodes aheterologous polypeptide.
 51. The method of claim 44 or 48, wherein theneurological disease or disorder is selected from the group consistingof Alzheimer's disease, Parkinson's disease, Huntington's disease,Pick's disease, Kuf's disease, Lewy body disease, neurofibrillarytangles, Rosenthal fibers, Mallory's hyaline, senile dementia,myasthenia gravis, Gilles de la Tourette's syndrome, multiple sclerosis(MS), amyotrophic lateral sclerosis (ALS), progressive supranuclearpalsy (PSP), epilepsy, Creutzfeldt-Jakob disease, deamess-dytoniasyndrome, Leigh syndrome, Leber hereditary optic neuropathy (LHON),parkinsonism, dystonia, motor neuron disease, neuropathy-ataxia andretinitis pimentosa (NARP), maternal inherited Leigh syndrome (MILS),Friedreich ataxia, hereditary spastic paraplegia, Mohr-Tranebjaergsyndrome, Wilson disease, sporatic Alzheimer's disease, sporadicamyotrophic lateral sclerosis, sporadic Parkinson's disease, autonomicfunction disorders, hypertension, sleep disorders, neuropsychiatricdisorders, depression, schizophrenia, schizoaffective disorder,korsakoff s psychosis, mania, anxiety disorders, phobic disorder,learning or memory disorders, amnesia or age-related memory loss,attention deficit disorder, dysthymic disorder, major depressivedisorder, obsessive-compulsive disorder, psychoactive substance usedisorders, panic disorder, bipolar affective disorder, severe bipolaraffective (mood) disorder (BP—I), migraines, hyperactivity and movementdisorders.
 52. The method of claim 44 or 48, wherein the neurologicaldisease or disorder is Parkinson's disease.
 53. The method of claim 44or 48, wherein the subject is human.
 54. The method of claim 44 or 48,wherein said modulator is administered in a pharmaceutically acceptableformulation.
 55. The method of claim 44 or 48, wherein the PGC-1αmodulator modulates mitochondrial function.
 56. The method of claim 55,wherein mitochondrial function is modulated in the brain.
 57. The methodof claim 44 or 48, wherein the PGC-1α modulator modulates lesionformation in the brain.
 58. The method of claim 44 or 48, wherein thePGC-1α modulator modulates neurite growth.